Dual Targeting siRNA  Agents

ABSTRACT

The invention relates to dual targeting siRNA agents targeting a PCSK9 gene and a second gene, and methods of using dual targeting siRNA agents to inhibit expression of PCSK9 and to treat PCSK9 related disorders, e.g., hyperlipidemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/521,422, filed Jul. 24, 2019, pending, which is a continuation ofU.S. application Ser. No. 15/724,175, filed Oct. 3, 2017, pending, whichis a continuation of U.S. application Ser. No. 14/885,342, filed Oct.16, 2015, (abandoned) which is a continuation of U.S. application Ser.No. 13/497,226, with a 371(c) filing date of Oct. 10, 2012, now U.S.Pat. No. 9,187,746, issued Nov. 17, 2015, which is a National Stage ofInternational Application No. PCT/US2010/049868, filed Sep. 22, 2010,which claims the benefit of U.S. Provisional Application No. 61/244,859,filed Sep. 22, 2009, and claims the benefit of U.S. ProvisionalApplication No. 61/313,584, filed Mar. 12, 2010, all of which are herebyincorporated in their entirety by reference.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing with 4166 sequencessubmitted electronically as a text file namedAYL108C4_sequencelisting.txt, created on Feb. 20, 2020, with a size of1,351,680 bytes. The sequence listing is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a composition of two covalently linked siRNAs,e.g., a dual targeting siRNA agent. At least one siRNA is a dsRNA thattargets a PCSK9 gene. The covalently linked siRNA agent is used inmethods of inhibition of PCSK9 gene expression and methods of treatmentof pathological conditions associated with PCSK9 gene expression, e.g.,hyperlipidemia.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of thesubtilisin serine protease family. The other eight mammalian subtilisinproteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6,PACE4, PC7, and S1P/SKI-1) are proprotein convertases that process awide variety of proteins in the secretory pathway and play roles indiverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol.24, 1-22, Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah,N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17,1215-1227, and Zhou, A., (1999) J Biol. Chem. 274, 20745-20748). PCSK9has been proposed to play a role in cholesterol metabolism. PCSK9 mRNAexpression is down-regulated by dietary cholesterol feeding in mice(Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated bystatins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc.Biol. 24, 1454-1459), and up-regulated in sterol regulatory elementbinding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc.Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterolbiosynthetic enzymes and the low-density lipoprotein receptor (LDLR).Furthermore, PCSK9 missense mutations have been found to be associatedwith a form of autosomal dominant hypercholesterolemia (Hchola3)(Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M.,(2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65,419-422). PCSK9 may also play a role in determining LDL cholesterollevels in the general population, because single-nucleotidepolymorphisms (SNPs) have been associated with cholesterol levels in aJapanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).

Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseasesin which patients exhibit elevated total and LDL cholesterol levels,tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) JClin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessiveform, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C.,(2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDLuptake by the liver. ADH may be caused by LDLR mutations, which preventLDL uptake, or by mutations in the protein on LDL, apolipoprotein B,which binds to the LDLR. ARH is caused by mutations in the ARH proteinthat are necessary for endocytosis of the LDLR-LDL complex via itsinteraction with clathrin. Therefore, if PCSK9 mutations are causativein Hchola3 families, it seems likely that PCSK9 plays a role inreceptor-mediated LDL uptake.

Overexpression studies point to a role for PCSK9 in controlling LDLRlevels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc.Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J.Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279,50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9for 3 or 4 days in mice results in elevated total and LDL cholesterollevels; this effect is not seen in LDLR knockout animals (Maxwell, K. N.(2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al.(2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol.Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in asevere reduction in hepatic LDLR protein, without affecting LDLR mRNAlevels, SREBP protein levels, or SREBP protein nuclear to cytoplasmicratio.

Loss of function mutations in PCSK9 have been designed in mouse models(Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in humanindividuals (Cohen et al. (2005) Nature Genetics 37:161-165). In bothcases loss of PCSK9 function lead to lowering of total and LDLccholesterol. In a retrospective outcome study over 15 years, loss of onecopy of PCSK9 was shown to shift LDLc levels lower and to lead to anincreased risk-benefit protection from developing cardiovascular heartdisease (Cohen et al., (2006) N Engl. J. Med., 354:1264-1272).

X-box binding protein 1 (XBP-1) is a basic leucine zipper transcriptionfactor that is involved in the cellular unfolded protein response (UPR).XBP-1 is known to be active in the endoplasmic reticulum (ER). The ERconsists of a system of folded membranes and tubules in the cytoplasm ofcells. Proteins and lipids are manufactured and processed in the ER.When unusual demands are placed on the ER, “ER stress” occurs. ER stresscan be triggered by a viral infection, gene mutations, exposure totoxins, aggregation of improperly folded proteins or a shortage ofintracellular nutrients. The result can be Type II diabetes, metabolicsyndrome, a neurological disorder or cancer.

Two XBP-1 isoforms are known to exist in cells: spliced XBP-1S andunspliced XBP-1U. Both isoforms of XBP-1 bind to the 21-bpTax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1)long terminal repeat (LTR) in vitro and transactivate HTLV-1transcription. HTLV-1 is associated with a rare form of blood dyscrasiaknown as Adult T-cell Leukemia/lymphoma (ATLL) and a myelopathy,tropical spastic paresis.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

A description of siRNA targeting PCSK9 can be found in U.S. patentapplication Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No.7,605,251) and International Patent Application No. PCT/US2007/068655filed May 10, 2007 (published as WO 2007/134161). Additional disclosurecan be found in U.S. patent application Ser. No. 12/478,452 filed Jun.4, 2009 (published as US 2010/0010066) and International PatentApplication No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO2009/134487).

A description of siRNA targeting XPB-1 can be found in U.S. patentapplication Ser. No. 12/425,811 filed on Apr. 17, 2009 and published asUS 2009-0275638.

Dual targeting siRNAs can be found in International patent applicationpublication no. WO/2007/091269.

SUMMARY OF THE INVENTION

Described herein are dual targeting siRNA agent in which a first siRNAtargeting PCSK9 is covalently joined to a second siRNA targeting a geneimplicated in cholesterol metabolism, e.g., XBP-1. The two siRNAs arecovalently linked via, e.g., a disulfide linker.

Accordingly one aspect of the invention is a dual targeting siRNA agenthaving a first dsRNA targeting a PCSK9 gene and a second dsRNA targetinga second gene, wherein the first dsRNA and the second dsRNA are linkedwith a covalent linker. The second gene is can be, e.g., XBP-1, PCSK9,PCSK5, ApoC3, SCAP, or MIG12. In one embodiment, the second gene isXBP-1. Each dsRNA is 30 nucleotides or less in length. In general, eachstrand of each dsRNA is 19-23 bases in length.

In one embodiment, the dual targeting siRNA agent comprising a firstdsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038targeting an XBP-1 gene, wherein AD-10792 sense strand and AD-18038sense strand are covalently linked with a disulfide linker.

The first dsRNA of the dual targeting siRNA agent targets a PCSK9 gene.In one aspect, the first dsRNA includes at least 15 contiguousnucleotides of an antisense strand of one of Tables 1, 2, or 4-8, orincludes an antisense strand of one of Tables 1, 2, or 4-8, or includesa sense strand and an antisense strand of one of Tables 1, 2, or 4-8.The first dsRNA can be AD-9680 or AD-10792.

In some embodiments, the second dsRNA target XBP-1. In one aspect, thesecond dsRNA includes at least 15 contiguous nucleotides of an antisensestrand of one of Tables 3 or 9-13, or includes an antisense strand ofone of Tables 3 or 9-13, or includes a sense strand and an antisensestrand of one of Tables 3 or 9-13. For example, the second dsRNA can beAD-18038.

Either the first and second dsRNA can include at least one modifiednucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, a terminal nucleotide linked toa cholesteryl derivative or dodecanoic acid bisdecylamide group, a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide. In someembodiments, the first and second dsRNAs include “endo-light”modification with 2′-O-methyl modified nucleotides and nucleotidescomprising a 5′-phosphorothioate group.

The first and second dsRNAs are linked with a covalent linker. In someembodiments, the linker is a disulfide linker. Various combinations ofstrands can be linked, e.g., the first and second dsRNA sense strandsare covalently linked or, e.g., the first and second dsRNA antisensestrands are covalently linked. In some embodiments, any of the dualtargeting siRNA agents of the invention include a ligand.

Also included in the invention are isolated cells having and vectorsencoding the dual targeting siRNA agent described herein.

In one aspect, administration of the dual targeting siRNA agent to acell inhibits expression of the PCSK9 gene and the second gene at alevel equivalent to inhibition of expression of both genes usingadministration of each siRNA individually. In another aspect,administration of the dual targeting siRNA agent to a subject results ina greater reduction of total serum cholesterol that that obtained byadministration of each siRNA alone.

The invention also includes a pharmaceutical composition comprising thedual targeting siRNA agents described herein and a pharmaceuticalcarrier. In one embodiment, the pharmaceutical carrier is a lipidformulation, e.g., a lipid formulation including cationic lipid DLinDMAor cationic lipid XTC. Examples of lipid formulations described in (butnot limited to) Table A, below. The lipid formulation can beXTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.

Another aspect of the invention includes methods of using the dualtargeting siRNA agents described herein. In one embodiment, theinvention is a method of inhibiting expression of the PCSK9 gene and asecond gene in a cell, the method comprising (a) introducing into thecell the any of the dual targeting siRNA agents and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of the PCSK9 gene and the second gene, therebyinhibiting expression of the PCSK9 gene and the second gene in the cell.

In another embodiment, the invention includes methods of treating adisorder mediated by PCSK9 expression with the step of administering toa subject in need of such treatment a therapeutically effective amountof the pharmaceutical compositions described herein. In one aspect, thedisorder is hyperlipidemia. In still another embodiment, the inventionincludes methods of reducing total serum cholesterol in a subjectcomprising administering to the subject a therapeutically effectiveamount of the pharmaceutical compositions described herein.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the effect on PCSK9 mRNA levels in primarymouse hepatocytes following treatment with a dual targeting siRNA,AD-23426. AD-23426 was as effective at reducing mRNA expression as eachsingle gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.Lipo2000: control transfection agent only FIG. 1B is a graph showing theeffect on XBP-1 mRNA levels in primary mouse hepatocytes followingtreatment with a dual targeting siRNA, AD-23426. AD-23426 was aseffective at reducing mRNA expression as each single gene target siRNA.AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: controltransfection agent only.

FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA levels inmice following treatment with a dual targeting siRNA, AD-23426. LNP09(lipid) formulated siRNA was administered to mice as described. AD-23426was as effective at reducing mRNA expression as each single gene targetsiRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.

FIG. 3 is a graph showing the effect on serum cholesterol levels in micefollowing treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid)formulated siRNA was administered to mice as described. AD-23426 wasmore effective at reducing serum cholesterol compared to each singlegene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.

FIG. 4A is a graph showing the effect on IFN-α in human PBMC followingtreatment with a dual targeting siRNA, AD-23426. FIG. 4B is a graphshowing the effect on TNF-α in human PBMC following treatment with adual targeting siRNA, AD-23426. DOTAP and LNP09 (lipid) formulatedsiRNAs was administered huPBMC as described below. AD-23426 did notinduce IFN-α or TNF-α.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by the down regulation of the PCSK9 gene, such ashyperlipidemia, by using dual targeting siRNA to silence the PCSK9 gene.

The invention provides compositions and methods for inhibiting theexpression of the PCSK9 gene in a subject using two siRNA, e.g., a dualtargeting siRNA. The invention also provides compositions and methodsfor treating pathological conditions and diseases, such ashyperlipidemia, that can be modulated by down regulating the expressionof the PCSK9 gene.

The dual targeting siRNA agents target a PCSK9 gene and at least oneother gene. The other gene can be another region of the PCSK9 gene, orcan be another gene, e.g., XBP-1.

The dual targeting siRNA agents have the advantage of lower toxicity,lower off-target effects, and lower effective concentration compared toindividual siRNAs.

The use of the dual targeting siRNA dsRNAs enables the targeteddegradation of an mRNA that is involved in the regulation of the LDLreceptor and circulating cholesterol levels. Using cell-based and animalassays it was demonstrated that inhibiting both a PCSK9 gene and anXBP-1 gene using a dual targeting siRNA is at least as effective atinhibiting their corresponding targets as the use of single siRNAs. Itwas also demonstrated that administration of a dual targeting siRNAresults in a synergistic lowering of total serum cholesterol. Thus,reduction of total serum cholesterol is enhanced with a dual targetingsiRNA compared to a single target siRNA.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. “T” and “dT” are used interchangeably herein and refer toa deoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The term “PCSK9” refers to the proprotein convertase subtilisin kexin 9gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). Examples ofmRNA sequences to PCSK9 include but are not limited to the following:human: NM_174936; mouse: NM_153565, and rat: NM_199253. Additionalexamples of PCSK9 mRNA sequences are readily available using, e.g.,GenBank.

The term “XBP-1” refers to—Box Protein 1, which is also known asTax-responsive element-binding protein 5, TREBS, and XBP2. XBP-1sequence can be found as NCBI GeneID:7494 and RefSeq ID number:NM_005080(human) and NM_013842 (mouse). A dsRNA featured in the invention cantarget a specific XBP-1 isoform, e.g., the spliced form (XBP-1S) or theunspliced form (XBP-1U), or a dsRNA featured in the invention can targetboth isoforms by binding to a common region of the mRNA transcript.

The term “PCSK5” refers to the Proprotein convertase subtilisin/kexintype 5 gene, mRNA or protein belonging to the subtilisin-like proproteinconvertase family.

The term “ApoC3” refers to the Apolipoprotein C-III protein gene, mRNAor protein, and is a very low density lipoprotein (VLDL).

The term “SCAP” refers to the SREBP cleavage-activating protein gene,mRNA or protein. SCAP is a regulatory protein that is required for theproteolytic cleavage of the sterol regulatory element binding protein(SREBP). Example of siRNA targeting SCAP are described in U.S. patentapplication Ser. No. 11/857,120, filed on Sep. 18, 2007, published as US20090093426. This application and the siRNA sequences described thereinare incorporated by reference for all purposes.

The term “MIG12” is a gene also known as TMSB10 and TB10 refers to thethymosin beta 10 gene. Example of siRNA targeting MIG12 are describedInternational patent application no. PCT/US10/25444, filed on Feb. 25,2010, published as WO/20XX This application and the siRNA sequencesdescribed therein are incorporated by reference for all purposes.

As used herein, the term “iRNA” refers to an agent that contains RNA andwhich mediates the targeted cleavage of an RNA transcript via anRNA-induced silencing complex (RISC) pathway. The term iRNA includessiRNA.

As described in more detail below, the term “siRNA” and “siRNA agent”refers to a dsRNA that mediates the targeted cleavage of an RNAtranscript via an RNA-induced silencing complex (RISC) pathway.

A “double-stranded RNA” or “dsRNA,” as used herein, refers to an RNAmolecule or complex of molecules having a hybridized duplex region thatcomprises two anti-parallel and substantially complementary nucleic acidstrands, which will be referred to as having “sense” and “antisense”orientations with respect to a target RNA.

The term “dual targeting siRNA agent” refers to a composition of twosiRNAs, e.g., two dsRNAs. One dsRNA includes an antisense strand with afirst region of complementarity to a first target gene, e.g., PCSK9. Thesecond dsRNA include an antisense strand with a second region ofcomplementarity to a second target gene. In some embodiments, the firstand second target genes are identical, e.g., both are PCSK9 and eachdsRNA targets a different region of PCSK9. In other embodiments, thefirst and second target genes are different, e.g., the first dsRNAtargets PCSK9 and the second dsRNA targets a different gene, e.g.,XBP-1.

“Covalent linker” refers to a molecule for covalently joining twomolecules, e.g., two dsRNAs. As described in more detail below, the termincludes, e.g., a nucleic acid linker, a peptide linker, and the likeand includes disulfide linkers.

The term “target gene” refers to a gene of interest, e.g., PCSK9 or asecond gene, e.g., XBP-1, targeted by an siRNA of the invention forinhibition of expression.

As described in more detail below, “target sequence” refers to acontiguous portion of the nucleotide sequence of an mRNA molecule formedduring the transcription of a target gene, including mRNA that is aproduct of RNA processing of a primary transcription product. The targetportion of the sequence will be at least long enough to serve as asubstrate for iRNA-directed cleavage at or near that portion. Forexample, the target sequence will generally be from 9-36 nucleotides inlength, e.g., 15-30 nucleotides in length, including all sub-rangestherebetween.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they may form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofthe target gene (e.g., an mRNA encoding PCSK9 or a second gene, e.g.,XBP-1). For example, a polynucleotide is complementary to at least apart of a PCSK9 mRNA if the sequence is substantially complementary to anon-interrupted portion of an mRNA encoding PCSK9.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art. Strictlyspeaking, a “ribonucleoside” includes a nucleoside base and a ribosesugar, and a “ribonucleotide” is a ribonucleoside with one, two or threephosphate moieties. However, the terms “ribonucleoside” and“ribonucleotide” can be considered to be equivalent as used herein. TheRNA can be modified in the nucleobase structure or in theribose-phosphate backbone structure, e.g., as described herein below.However, the molecules comprising ribonucleoside analogs or derivativesmust retain the ability to form a duplex. As non-limiting examples, anRNA molecule can also include at least one modified ribonucleosideincluding but not limited to a 2′-O-methyl modified nucleotide, anucleoside comprising a 5′ phosphorothioate group, a terminal nucleosidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoromodified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modifiednucleoside, morpholino nucleoside, a phosphoramidate or a non-naturalbase comprising nucleoside, or any combination thereof. Alternatively,an RNA molecule can comprise at least two modified ribonucleosides, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20 or more, up to the entirelength of the dsRNA molecule. The modifications need not be the same foreach of such a plurality of modified ribonucleosides in an RNA molecule.In one embodiment, modified RNAs contemplated for use in methods andcompositions described herein are peptide nucleic acids (PNAs) that havethe ability to form the required duplex structure and that permit ormediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside.In such an instance, an iRNA agent can comprise one or moredeoxynucleosides, including, for example, a deoxynucleoside overhang(s),or one or more deoxynucleosides within the double stranded portion of adsRNA. However, it is self evident that under no circumstances is adouble stranded DNA molecule encompassed by the term “iRNA.”

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) may be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA. One or moreof the nucleotides in the overhang can be replaced with a nucleosidethiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA or a plasmidfrom which an iRNA is transcribed. SNALPs are described, e.g., in U.S.Patent Application Publication Nos. 20060240093, 20070135372, and inInternational Application No. WO 2009082817. These applications areincorporated herein by reference in their entirety.

“Introducing into a cell,” when referring to an iRNA, means facilitatingor effecting uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. The meaning of this term is not limited to cells invitro; an iRNA may also be “introduced into a cell,” wherein the cell ispart of a living organism. In such an instance, introduction into thecell will include the delivery to the organism. For example, for in vivodelivery, iRNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be by a beta-glucan deliverysystem, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Publication No. 2005/0281781, which are herebyincorporated by reference in their entirety. In vitro introduction intoa cell includes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or known inthe art.

As used herein, the term “modulate the expression of,” refers to at anleast partial “inhibition” or partial “activation” of target geneexpression in a cell treated with an iRNA composition as describedherein compared to the expression of the target gene in an untreatedcell.

The terms “activate,” “enhance,” “up-regulate the expression of,”“increase the expression of,” and the like, in so far as they refer to atarget gene, herein refer to the at least partial activation of theexpression of a target gene, as manifested by an increase in the amountof target mRNA, which may be isolated from or detected in a first cellor group of cells in which a target gene is transcribed and which has orhave been treated such that the expression of a target gene isincreased, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells).

In one embodiment, expression of a target gene is activated by at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administrationof an iRNA as described herein. In some embodiments, a target gene isactivated by at least about 60%, 70%, or 80% by administration of aniRNA featured in the invention. In some embodiments, expression of atarget gene is activated by at least about 85%, 90%, or 95% or more byadministration of an iRNA as described herein. In some embodiments, thetarget gene expression is increased by at least 1-fold, at least 2-fold,at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold,at least 500-fold, at least 1000 fold or more in cells treated with aniRNA as described herein compared to the expression in an untreatedcell. Activation of expression by small dsRNAs is described, forexample, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42,and in US20070111963 and US2005226848, each of which is incorporatedherein by reference.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to a target gene, herein refer to the at least partialsuppression of the expression of a target gene, as manifested by areduction of the amount of target mRNA which may be isolated from ordetected in a first cell or group of cells in which a target gene istranscribed and which has or have been treated such that the expressionof target gene is inhibited, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to target geneexpression, e.g., the amount of protein encoded by a target gene, or thenumber of cells displaying a certain phenotype, e.g., lack of ordecreased cytokine production. In principle, target gene silencing maybe determined in any cell expressing target, either constitutively or bygenomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given iRNA inhibitsthe expression of the target gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of a target gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, or 55% by administration of an iRNA featured in the invention. Insome embodiments, a target gene is suppressed by at least about 60%,65%, 70%, 75%, or 80% by administration of an iRNA featured in theinvention. In some embodiments, a target gene is suppressed by at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA asdescribed herein.

As used herein in the context of target gene expression, the terms“treat,” “treatment,” and the like, refer to relief from or alleviationof pathological processes mediated by target expression. In the contextof the present invention insofar as it relates to any of the otherconditions recited herein below (other than pathological processesmediated by target expression), the terms “treat,” “treatment,” and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression or anticipatedprogression of such condition.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40% or more, and is preferably down toa level accepted as within the range of normal for an individual withoutsuch disorder.

As used herein, the phrase “therapeutically effective amount”” refers toan amount that provides a therapeutic benefit in the treatment ormanagement of pathological processes mediated by target gene expression,e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom ofpathological processes mediated target gene expression. The phrase“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the prevention of pathological processes mediatedby target gene expression or an overt symptom of pathological processesmediated by target gene expression. The specific amount that istherapeutically effective can be readily determined by an ordinarymedical practitioner, and may vary depending on factors known in theart, such as, for example, the type of pathological processes mediatedby target gene expression, the patient's history and age, the stage ofpathological processes mediated by target gene expression, and theadministration of other agents that inhibit pathological processesmediated by target gene expression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an iRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an iRNA effective to produce the intendedpharmacological or therapeutic result. For example, if a given clinicaltreatment is considered effective when there is at least a 10% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 10%reduction in that parameter.

The term “pharmaceutically carrier” refers to a carrier foradministration of a therapeutic agent, e.g., a dual targeting siRNAagent. Carriers are described in more detail below, and include lipidformulations, e.g., LNP09 and SNALP formulations.

Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are dual targeting siRNA agents, e.g., siRNAs thatinhibit the expression of a PCSK9 gene and a second gene. The dualtargeting siRNA agent includes two siRNA covalently linked via, e.g., adisulfide linker. The first siRNA targets a first region of a PCSK9gene. The second siRNA targets a second gene, e.g., XBP-1, or, e.g.,targets a second region of the PCSK9 gene.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, AppliedBiosystems, Inc. Further descriptions of synthesis are found below andin the examples.

Each siRNA is a dsRNA. A dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure underconditions in which the dsRNA will be used. One strand of a dsRNA (theantisense strand) includes a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of a target gene. The other strand (the sense strand)includes a region that is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions.

Where the duplex region is formed from two strands of a single molecule,the molecule can have a duplex region separated by a single strandedchain of nucleotides (herein referred to as a “hairpin loop”) betweenthe 3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure. The hairpin loop can comprise at least oneunpaired nucleotide; in some embodiments the hairpin loop can compriseat least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 20, at least 23 or more unpairednucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than a hairpin loop, the connecting structure is referredto as a “linker.”

Generally, the duplex structure of the siRNA, e.g., dsRNA, is between 15and 30 inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Considering a duplex between 9 and36 base pairs, the duplex can be any length in this range, for example,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range thereinbetween, including, but not limited to 15-30 base pairs, 15-26 basepairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 basepairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 basepairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 basepairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 basepairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 basepairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 basepairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 basepairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22base pairs.

The two siRNAs in the dual targeting siRNA agent can have duplex lengthsthat are identical or that differ.

The region of complementarity to the target sequence in an siRNA isbetween 15 and 30 inclusive, more generally between 18 and 25 inclusive,yet more generally between 19 and 24 inclusive, and most generallybetween 19 and 21 nucleotides in length, inclusive. In some embodiments,the dsRNA is between 15 and 20 nucleotides in length, inclusive, and inother embodiments, the dsRNA is between 25 and 30 nucleotides in length,inclusive. The region of complementarity can be 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Asnon-limiting examples, the target sequence can be from 15-30nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides,15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides,18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides,19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides,21-23 nucleotides, or 21-22 nucleotides. In some embodiments the targetsequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

The two siRNAs in the dual targeting siRNA agent can have regions ofcomplementarity that are identical in length or that differ in length.

Any of the dsRNA, e.g., siRNA as described herein may include one ormore single-stranded nucleotide overhangs. In one embodiment, at leastone end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4,or 1 or 2 or 3 or 4 nucleotides. dsRNAs having at least one nucleotideoverhang have unexpectedly superior inhibitory properties relative totheir blunt-ended counterparts. Generally, the single-stranded overhangis located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate. The two siRNAs inthe dual targeting siRNA agent can have different or identical overhangsas described by location, length, and nucleotide.

The dual targeting siRNA agent includes at least a first siRNA targetinga first region of a PCSK9 gene. In one embodiment, a PCSK9 gene is ahuman PCSK9 gene. In another embodiment the PCSK9 gene is a mouse or arat PCSK9 gene. Exemplary siRNA targeting PCSK9 are described in U.S.patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S.Pat. No. 7,605,251) and International Patent Application No.PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161).Additional disclosure can be found in U.S. patent application Ser. No.12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) andInternational Patent Application No. PCT/US2009/032743 filed Jan. 30,2009 (published as WO 2009/134487). The sequences of the target, sense,and antisense strands are incorporated by reference for all purposes.

Tables 1, 2, and 4-8 disclose sequences of the target, sense strands,and antisense strands of PCSK9 targeting siRNA.

In one embodiment the first siRNA is AD-9680. The dsRNA AD-9680 targetsthe human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene(accession number NM_174936).

TABLE 1 AD-9680 siRNA sequences Table 1: AD-9680 Sequence 5′ to 3′SEQ ID NO: Target sequence UUCUAGACCUGUUUUGCUU 4142 Sense strandUUCUAGACCUGUUUUGCUU 4143 Sense strand, uucuAGAccuGuuuuGcuuTsT 4144modified Antisense strand AAGCAAAACAGGUCUAGAA 4145 Antisense strand,AAGcAAAAcAGGUCuAGAATsT 4146 modified

In another embodiment, the first siRNA is AD-10792. The dsRNA AD-10792targets the PCSK9 gene at nucleotides 1091-1109 of a human PCSK9 gene(accession number NM_174936). AD-10792 is also complementary to rodentPCSK9.

TABLE 2 AD-10792 siRNA sequences SEQ Table 2: AD-10792 Sequence 5′ to 3′ID NO: Target sequence GCCUGGAGUUUAUUCGGAA 4147 Sense strandGCCUGGAGUUUAUUCGGAA 4148 Sense strand, GccuGGAGuuuAuucGGAATsT 4149modified Antisense strand UUCCGAAUAAACUCCAGGC 4150 Antisense strand,UUCCGAAuAAACUCcAGGCTsT 4151 modified

The second siRNA of the dual targeting siRNA agent targets a secondgene. In one embodiment, the second gene is PCSK9, and the second siRNAtarget a region of PCSK9 that is different from the region targeted bythe first siRNA.

Alternatively, the second siRNA targets a different second gene.Examples include genes that interact with PCSK9 and/or are involved withlipid metabolism or cholesterol metabolism. For example, the secondtarget gene can be XBP-1, PCSK5, ApoC3, SCAP, MIG12, HMG CoA Reductase,or IDOL (Inducible Degrader of the LDLR) and the like. In oneembodiment, the second gene is a human gene. In another embodiment thesecond gene is a mouse or a rat gene.

In one embodiment, the second siRNA targets the XBP-1 gene. ExemplarysiRNA targeting XBP-1 can be found in U.S. patent application Ser. No.12/425,811 filed Apr. 17, 2009 (published as US 2009-0275638). Thesequences of the target, sense, and antisense strands are incorporatedby reference for all purposes.

Tables 3 and 9-13 disclose sequences of the target, sense strands, andantisense strands of XBP-1 targeting siRNA.

In one embodiment the first siRNA is AD-18038. The dsRNA AD-18038targets the human XBP-1 gene at nucleotides 896-914 of a human XBP-1gene (accession number NM_001004210).

TABLE 3 AD-18038 siRNA sequences Table 3: AD-18038 Sequence 5′ to 3′SEQ ID NO: Target sequence CACCCUGAAUUCAUUGUCU 4153 Sense strandCACCCUGAAUUCAUUGUCU 4154 Sense strand, modified cAcccuGAAuucAuuGucudTsdT4155 Antisense strand AGACAAUGAAUUCAGGGUG 4156Antisense strand, modified AGAcAAUGAAUUcAGGGUGdTsdT 4157

Additional dsRNA

A dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from one of the sequences in Tables 1-13,and differing in their ability to inhibit the expression of a targetgene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated according to theinvention.

In addition, the RNAs provided in Tables 1-13 identify a site in thetarget gene transcript that is susceptible to RISC-mediated cleavage. Assuch, the present invention further features iRNAs that target withinone of such sequences. As used herein, an iRNA is said to target withina particular site of an RNA transcript if the iRNA promotes cleavage ofthe transcript anywhere within that particular site. Such an iRNA willgenerally include at least 15 contiguous nucleotides from one of thesequences provided herein coupled to additional nucleotide sequencestaken from the region contiguous to the selected sequence in a targetgene.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that mayserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, above represent effectivetarget sequences, it is contemplated that further optimization ofinhibition efficiency can be achieved by progressively “walking thewindow” one nucleotide upstream or downstream of the given sequences toidentify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 1-13, further optimization could be achieved by systematicallyeither adding or removing nucleotides to generate longer or shortersequences and testing those and sequences generated by walking a windowof the longer or shorter size up or down the target RNA from that point.Again, coupling this approach to generating new candidate targets withtesting for effectiveness of iRNAs based on those target sequences in aninhibition assay as known in the art or as described herein can lead tofurther improvements in the efficiency of inhibition. Further still,such optimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes, etc.) as an expressioninhibitor.

An iRNA as described in Tables 1-13 can contain one or more mismatchesto the target sequence. In one embodiment, an iRNA as described inTables 1-13 contains no more than 3 mismatches. If the antisense strandof the iRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the iRNA contains mismatchesto the target sequence, it is preferable that the mismatch be restrictedto be within the last 5 nucleotides from either the 5′ or 3′ end of theregion of complementarity. For example, for a 23 nucleotide iRNA agentRNA strand which is complementary to a region of a PCSK9 gene, the RNAstrand generally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of a PCSK9gene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of a PCSK9 gene is important, especially if theparticular region of complementarity in a PCSK9 gene is known to havepolymorphic sequence variation within the population.

Covalent Linkage

The dual targeting siRNA agents of the invention include two siRNAsjoined via a covalent linker. Covalent linkers are well-known to one ofskill in the art and include, e.g., a nucleic acid linker, a peptidelinker, and the like.

The covalent linker joins the two siRNAs. The covalent linker can jointwo sense strands, two antisense strands, one sense and one antisensestrand, two sense strands and one antisense strand, two antisensestrands and one sense strand, or two sense and two antisense strands.

The covalent linker can include RNA and/or DNA and/or a peptide. Thelinker can be single stranded, double stranded, partially singlestrands, or partially double stranded. In some embodiments the linkerincludes a disulfide bond. The linker can be cleavable or non-cleavable.

The covalent linker can be, e.g.,dTsdTuu=(5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate);rUsrU (a thiophosphate linker:5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate); an rUrU linker;dTsdTaa (aadTsdT,5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-adenyl-3′-phosphate-5′-adenyl-3′-phosphate);dTsdT (5′-2′deoxythyrnidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate);dTsdTuu=uudTsdT=5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate.

The covalent linker can be a polyRNA, such aspoly(5′-adenyl-3′-phosphate—AAAAAAAA) orpoly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate—CUCUCUCU)), e.g.,X_(n) single stranded poly RNA linker wherein n is an integer from 2-50inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive.Modified nucleotides or a mixture of nucleotides can also be present insaid polyRNA linker. The covalent linker can be a polyDNA, such aspoly(5′-2′deoxythymidyl-3′-phosphate—TTTTTTTT), e.g., wherein n is aninteger from 2-50 inclusive, preferable 4-15 inclusive, most preferably7-8 inclusive. Modified nucleotides or a mixture of nucleotides can alsobe present in said polyDNA linker. a single stranded polyDNA linkerwherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive,most preferably 7-8 inclusive. Modified nucleotides or a mixture ofnucleotides can also be present in said polyDNA linker.

The covalent linker can include a disulfide bond, optionally abis-hexyl-disulfide linker. In one embodiment, the disulfide linker is

The covalent linker can include a peptide bond, e.g., include aminoacids. In one embodiment, the covalent linker is a 1-10 amino acid longlinker, preferably comprising 4-5 amino acids, optionallyX-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.

The covalent linker can include HEG, a hexaethylenglycol linker.

Modifications

In yet another embodiment, at least one of the siRNA of the dualtargeting siRNA agent is chemically modified to enhance stability orother beneficial characteristics. The nucleic acids featured in theinvention may be synthesized and/or modified by methods well establishedin the art, such as those described in “Current protocols in nucleicacid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,Inc., New York, N.Y., USA, which is hereby incorporated herein byreference. Modifications include, for example, (a) end modifications,e.g., 5′ end modifications (phosphorylation, conjugation, invertedlinkages, etc.) 3′ end modifications (conjugation, DNA nucleotides,inverted linkages, etc.), (b) base modifications, e.g., replacement withstabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, removal of bases (abasic nucleotides),or conjugated bases, (c) sugar modifications (e.g., at the 2′ positionor 4′ position) or replacement of the sugar, as well as (d) backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of RNA compounds useful inthis invention include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the modified RNA willhave a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C_(m)alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C_(m) lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2′-docosanoyl-uridine-3′-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in U.S. Provisional PatentApplication No. 61/223,665 (“the '665 application”), filed Jul. 7, 2009,entitled “Oligonucleotide End Caps” and International patent applicationno. PCT/US10/41214, filed Jul. 7, 2010.

Ligands

Another modification of the RNA of an iRNA featured in the inventioninvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one ligand, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:4158). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:4159)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:4160)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:4161))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit is a cell targeting peptidesuch as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an dsRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver a iRNA agent to a tumor cell expressing αvβ3 (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; each of which is herein incorporated by reference.

Chimeras

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or“chimeras,” in the context of this invention, are iRNA compounds,preferably dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These iRNAs typically contain at leastone region wherein the RNA is modified so as to confer upon the iRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the iRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of iRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter iRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Non-ligand groups

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved ina number of different ways. In vivo delivery can be performed directlyby administering a composition comprising an iRNA, e.g. a dsRNA, to asubject. Alternatively, delivery can be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can beadapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). However, there are threefactors that are important to consider in order to successfully deliveran iRNA molecule in vivo: (a) biological stability of the deliveredmolecule, (2) preventing non-specific effects, and (3) accumulation ofthe delivered molecule in the target tissue. The non-specific effects ofan iRNA can be minimized by local administration, for example by directinjection or implantation into a tissue (as a non-limiting example, atumor) or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that may otherwise beharmed by the agent or that may degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, the dsRNAs of the invention can be expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as aninverted repeat joined by a linker polynucleotide sequence such that thedsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct maybe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an iRNA can be used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding an iRNA are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdrl gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of iRNAs.Adenoviruses are especially attractive vehicles, e.g., for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the iRNA can be expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector having, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressingthe dsRNA featured in the invention, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479;5,139,941; International Patent Application No. WO 94/13788; andInternational Patent Application No. WO 93/24641, the entire disclosuresof which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing iRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dual targeting siRNA agent, as described herein, and apharmaceutically acceptable carrier. The pharmaceutical compositioncontaining the siRNA is useful for treating a disease or disorderassociated with the expression or activity of a target gene, such aspathological processes mediated by PCSK9 expression. Such pharmaceuticalcompositions are formulated based on the mode of delivery. One exampleis compositions that are formulated for systemic administration viaparenteral delivery, e.g., by intravenous (IV) delivery. Another exampleis compositions that are formulated for direct delivery into the brainparenchyma, e.g., by infusion into the brain, such as by continuous pumpinfusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of the target genes. Ingeneral, a suitable dose of siRNA will be in the range of 0.01 to 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at 0.01 mg/kg, 0.02 mg/kg, 0.03mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg,0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg,2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg,10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg per singledose.

The pharmaceutical composition may be administered once daily, or theiRNA may be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The effect of a single dose of siRNA on PCSK9 levels can be longlasting, such that subsequent doses are administered at not more than 3,4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by PCSK9 expression. Such models can be used for in vivotesting of iRNA, as well as for determining a therapeutically effectivedose. A suitable mouse model is, for example, a mouse containing atransgene expressing human PCSK9.

The present invention also includes pharmaceutical compositions andformulations that include the iRNA compounds featured in the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general, their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a dual targeting siRNA agent featured in theinvention is fully encapsulated in the lipid formulation, e.g., to forma nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or SNALP. As usedherein, the term “SNALP” refers to a stable nucleic acid-lipid particle,including SPLP. As used herein, the term “SPLP” refers to a nucleicacid-lipid particle comprising plasmid DNA encapsulated within a lipidvesicle. Nucleic acid-lipid particles, e.g., SNALPs, typically contain acationic lipid, a non-cationic lipid, and a lipid that preventsaggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs andSPLPs are extremely useful for systemic applications, as they exhibitextended circulation lifetimes following intravenous (i.v.) injectionand accumulate at distal sites (e.g., sites physically separated fromthe administration site). SPLPs include “pSPLP”, which include anencapsulated condensing agent-nucleic acid complex as set forth in PCTPublication No. WO 00/03683.

The particles of the present invention typically have a mean diameter ofabout 50 nm to about 150 nm, more typically about 60 nm to about 130 nm,more typically about 70 nm to about 110 nm, most typically about 70 nmto about 90 nm, and are substantially nontoxic. For example, the meandiameter of the particles can be about 50 nm, 55 nm, 60 nm, 65 nm, 70nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm,120 nm, 125 nm, 130 nm, 140 nm, 145 nm, or 150 nm.

In addition, the nucleic acids when present in the nucleic acid-lipidparticles of the present invention are resistant in aqueous solution todegradation with a nuclease. Nucleic acid-lipid particles and theirmethod of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567;5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. The lipid to dsRNA ratio can be about 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1,33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1,45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.

The nucleic acid lipid particles include a cationic lipid. The cationiclipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof, 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(XTC),(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyOdidodecan-2-ol(Tech G1, e.g., C12-200), or a mixture thereof.

The cationic lipid may comprise from about 10 mol % to about 70 mol % orabout 40 mol % of the total lipid present in the particle. The cationiclipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the totallipid present in the particle. The cationic lipid may comprise 57.1 mol% or 57.5 mol % of the total lipid present in the particle.

In one embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC) can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±nm and a 0.027 siRNA/Lipid Ratio.

The nucleic acid lipid particle generally includes a non-cationic lipid.The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof.

The non-cationic lipid may be from about 5 mol % to about 90 mol %,about 10 mol %, or about 58 mol % if cholesterol is included, of thetotal lipid present in the particle. The non-cationic lipid may be about5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %, 9 mol %, 10mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17mol %, 18 mol %, 19 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol %.

The nucleic acid lipid particle generally includes a conjugated lipid.The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (C12), aPEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid can be PEG-DMG(PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wtof 2000); PEG-DSG (PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG withavg mol wt of 2000); or PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of2000).

The conjugated lipid that prevents aggregation of particles may be from0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or20.0 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle. For example, the nucleicacid-lipid particle further includes cholesterol at about 5 mol %, 10mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45mol %, 50 mol %, 55 mol %, or 60 mol %. The nucleic acid-lipid particlecan include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5mol %, or 40 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which is hereinincorporated by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Exemplary Nucleic Acid Lipid Particles

Additional exemplary lipid-dsRNA formulations are as follows:

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Cationic Mol % ratios Lipid Lipid:siRNA ratio SNALP DLinDMADLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA~7:1S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4lipid:siRNA~7:1 LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5lipid:siRNA~6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5lipid:siRNA~11:1 LNP07 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5,lipid:siRNA~6:1 LNP08 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5,lipid:siRNA—11:1 LNP09 XTC XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA 10:1 LNP10 ALN100 ALN100/DSPC/Cholesterol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA 10:1 LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 C12-200C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP13XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/239,686, filed Sep. 3, 2009, and International patentapplication no. PCT/US 10/22614, filed Jan. 29, 2010, which are herebyincorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, which are hereby incorporated byreference.

ALN100, i.e., ALNY-100 comprising formulations are described, e.g.,International patent application number PCT/US09/63933, filed on Nov.10, 2009, which is hereby incorporated by reference.

C12-200, i.e., Tech G1 comprising formulations are described in U.S.Provisional Ser. No. 61/175,770, filed May 5, 2009, which is herebyincorporated by reference.

Synthesis of Cationic Lipids.

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention may be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below.

Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents may be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In one embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A; XTC is a cationic lipidof formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring.

In general, the lipid of formula A above may be made by the followingReaction Schemes 1 or 2, wherein all substituents are as defined aboveunless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R₃ and R₄ are independentlylower alkyl or R₃ and R₄ can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3 Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO₄and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3,[M+NH₄+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

General Synthesis of Nucleic Acid Lipid Particles

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Other Formulations

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants may beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene olyel ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants:

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of iRNAs through the mucosa is enhanced.In addition to bile salts and fatty acids, these penetration enhancersinclude, for example, sodium lauryl sulfate, polyoxyethylene-9-laurylether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92); and perfluorochemical emulsions, such asFC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty Acids:

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile Salts:

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents:

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-Chelating Non-Surfactants:

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers include, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lotto et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or morebiologic agents which function by a non-RNAi mechanism. Examples of suchbiologics include, biologics that target one or more of PD-1, PD-L1, orB7-H1 (CD80) (e.g., monoclonal antibodies against PD-1, PD-L1, orB7-H1), or one or more recombinant cytokines (e.g., IL6, IFN-γ, andTNF).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dualtargeting siRNAs featured in the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by PCSK9 expression. In any event, theadministering physician can adjust the amount and timing of iRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods Using Dual Targeting siRNAs

In one aspect, the invention provides use of a dual targeting siRNAagent for inhibiting the expression of the PCSK9 gene in a mammal. Themethod includes administering a composition of the invention to themammal such that expression of the target PCSK9 gene is decreased. Insome embodiments, PCSK9 expression is decreased for an extendedduration, e.g., at least one week, two weeks, three weeks, or four weeksor longer. For example, in certain instances, expression of the PCSK9gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, or 50% by administration of a dual targeting siRNA agentdescribed herein. In some embodiments, the PCSK9 gene is suppressed byat least about 60%, 70%, or 80% by administration of the dual targetingsiRNA agent. In some embodiments, the PCSK9 gene is suppressed by atleast about 85%, 90%, or 95% by administration of the double-strandedoligonucleotide.

The methods and compositions described herein can be used to treatdiseases and conditions that can be modulated by down regulating PCSK9gene expression. For example, the compositions described herein can beused to treat hyperlipidemia and other forms of lipid imbalance such ashypercholesterolemia, hypertriglyceridemia and the pathologicalconditions associated with these disorders such as heart and circulatorydiseases

Therefore, the invention also relates to the use of a dual targetingsiRNA agent for the treatment of a PCSK9-mediated disorder or disease.For example, a dual targeting siRNA agent is used for treatment of ahyperlipidemia.

The effect of the decreased PCSK9 gene preferably results in a decreasein LDLc (low density lipoprotein cholesterol) levels in the blood, andmore particularly in the serum, of the mammal. In some embodiments, LDLclevels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or60%, or more, as compared to pretreatment levels.

The method includes administering a dual targeting siRNA agent to thesubject to be treated. When the organism to be treated is a mammal suchas a human, the composition can be administered by any means known inthe art including, but not limited to oral or parenteral routes,including intravenous, intramuscular, subcutaneous, transdermal, andairway (aerosol) administration. In some embodiments, the compositionsare administered by intravenous infusion or injection.

The method includes administering a dual targeting siRNA agent, e.g., adose sufficient to depress levels of PCSK9 mRNA for at least 5, morepreferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally,administering a second single dose of dsRNA, wherein the second singledose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30or 40 days after the first single dose is administered, therebyinhibiting the expression of the PCSK9 gene in a subject.

In one embodiment, doses of dual targeting siRNA agent are administerednot more than once every four weeks, not more than once every threeweeks, not more than once every two weeks, or not more than once everyweek. In another embodiment, the administrations can be maintained forone, two, three, or six months, or one year or longer.

In another embodiment, administration can be provided when Low DensityLipoprotein cholesterol (LDLc) levels reach or surpass a predeterminedminimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200mg/dL, 300 mg/dL, or 400 mg/dL.

In general, the dual targeting siRNA agent does not activate the immunesystem, e.g., it does not increase cytokine levels, such as TNF-alpha orIFN-alpha levels. For example, when measured by an assay, such as an invitro PBMC assay, such as described herein, the increase in levels ofTNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cellstreated with a control dsRNA, such as a dsRNA that does not targetPCSK9.

For example, a subject can be administered a therapeutic amount of dualtargeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, or 2.5 mg/kg dsRNA. The dual targeting siRNA agent can beadministered by intravenous infusion over a period of time, such as overa 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. Theadministration is repeated, for example, on a regular basis, such asbiweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration biweekly for three months, administration can berepeated once per month, for six months or a year or longer.Administration of the dual targeting siRNA agent can reduce PCSK9levels, e.g., in a cell, tissue, blood, urine or other compartment ofthe patient by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80% or at least 90% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction, or forelevated lipid levels or blood pressure. In another example, the patientcan be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given dual targeting siRNA agent drug orformulation of that drug can also be judged using an experimental animalmodel for the given disease as known in the art. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant reduction in a marker or symptom is observed.

Additional Agents

In further embodiments, administration of a dual targeting siRNA agentis administered in combination an additional therapeutic agent. The dualtargeting siRNA agent and an additional therapeutic agent can beadministered in combination in the same composition, e.g., parenterally,or the additional therapeutic agent can be administered as part of aseparate composition or by another method described herein.

Examples of additional therapeutic agents include those known to treatan agent known to treat a lipid disorders, such as hypercholesterolemia,atherosclerosis or dyslipidemia. For example, a dual targeting siRNAagent featured in the invention can be administered with, e.g., anHMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acidsequestrant, niacin, an antiplatelet agent, an angiotensin convertingenzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartanpotassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterolacetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor,a cholesterol ester transfer protein (CETP) inhibitor, a microsomaltriglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator,a bile acid modulator, a peroxisome proliferation activated receptor(PPAR) agonist, a gene-based therapy, a composite vascular protectant(e.g., AGI-1067, from Atherogenics), a glycoprotein inhibitor, aspirinor an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, fromShionogi), a squalene synthase inhibitor, or a monocyte chemoattractantprotein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitorsinclude atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl),pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo'sMevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, BoehringerIngelheim's Denan, Banyu's Lipovas), lovastatin (Merck'sMevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma'sLiposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa'sCranoc, Solvay's Digaril), cerivastatin (Bayer'sLipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca'sCrestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical,Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g.,bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol),clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier'sLipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics),gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate(Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrantsinclude, e.g., cholestyramine (Bristol-Myers Squibb's Questran® andQuestran Light™), colestipol (e.g., Pharmacia's Colestid), andcolesevelam (Genzyme/Sankyo's WelChol′). Exemplary niacin therapiesinclude, e.g., immediate release formulations, such as Aventis' Nicobid,Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit.Niacin extended release formulations include, e.g., Kos Pharmaceuticals'Niaspan and Upsher-Smith's Slo-Niacin. Exemplary antiplatelet agentsinclude, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel(Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine(e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Otheraspirin-like compounds useful in combination with a dsRNA targetingPCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) andPamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplaryangiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g.,Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplaryacyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g.,avasimibe (Pfizer), eflucimibe (BioM{circumflex over (ε)}rieux PierreFabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe(Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer).Exemplary CETP inhibitors include, e.g., Torcetrapib (also calledCP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (AvantImmunotherapeutics). Exemplary microsomal triglyceride transfer protein(MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757(Janssen), and CP-346086 (Pfizer). Other exemplary cholesterolmodulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027(Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulatorsinclude, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British TechnologyGroup), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), andAZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activatedreceptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242)(AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson),GW-409544 (Ligand Pharniaceuticals/GlaxoSmithKline), GW-501516 (LigandPharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and EliLilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674(Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin).Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec),ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics),and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte,Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include,e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban(Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, e.g., BMS-1884941(Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer),CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitoris, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agentBO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivativeNyclin (Yamanouchi Pharmacuticals) are also appropriate foradministering in combination with a dsRNA featured in the invention.Exemplary combination therapies suitable for administration with a dsRNAtargeting PCSK9 include, e.g., advicor (Niacin/lovastatin from KosPharmaceuticals), amlodipine/atorvastatin (Pfizer), andezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treatinghypercholesterolemia, and suitable for administration in combinationwith a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev®Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets(Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets(AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis),fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodiumLipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules(Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets(Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott),fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-PloughPharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo),colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia®Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor®Tablets (Merck).

In one embodiment, a dual targeting siRNA agent is administered incombination with an ezetimibe/simvastatin combination (e.g., Vytorin®(Merck/Schering-Plough Pharmaceuticals)).

In one embodiment, the dual targeting siRNA agent is administered to thepatient, and then the additional therapeutic agent is administered tothe patient (or vice versa). In another embodiment, the dual targetingsiRNA agent and the additional therapeutic agent are administered at thesame time.

In another aspect, the invention features, a method of instructing anend user, e.g., a caregiver or a subject, on how to administer a dualtargeting siRNA agent described herein. The method includes, optionally,providing the end user with one or more doses of the dual targetingsiRNA agent, and instructing the end user to administer the dualtargeting siRNA agent on a regimen described herein, thereby instructingthe end user.

Identification of Patients

In one aspect, the invention provides a method of treating a patient byselecting a patient on the basis that the patient is in need of LDLlowering, LDL lowering without lowering of HDL, ApoB lowering, or totalcholesterol lowering. The method includes administering to the patient adual targeting siRNA agent in an amount sufficient to lower thepatient's LDL levels or ApoB levels, e.g., without substantiallylowering HDL levels.

Genetic predisposition plays a role in the development of target geneassociated diseases, e.g., hyperlipidemia. Therefore, a patient in needof a dual targeting siRNA agent can be identified by taking a familyhistory, or, for example, screening for one or more genetic markers orvariants. A healthcare provider, such as a doctor, nurse, or familymember, can take a family history before prescribing or administering adual targeting siRNA agent. For example, a DNA test may also beperformed on the patient to identify a mutation in the PCSK9 gene,before a PCSK9 dsRNA is administered to the patient.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG, 500Å, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN₆-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichis used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes is used. The activator is 5-ethyl thiotetrazole(0.75M, American International Chemicals); for the PO-oxidationiodine/water/pyridine is used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite are purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55°C. The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to −30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

iRNA Preparation

For the general preparation of iRNA, equimolar amounts of sense andantisense strand are heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex is confirmed by HPLCanalysis.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table B.

TABLE B Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide (s) A adenosine Ccytidine G guanosine U uridine N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u2′-O-methyluridine dT, T 2′-deoxythymidine s phosphorothioate linkage

Example 2. PCSK9 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using PCSK9 siRNA canbe found in detail in U.S. patent application Ser. No. 11/746,864 filedon May 10, 2007 (now U.S. Pat. No. 7,605,251) and International PatentApplication No. PCT/US2007/068655 filed May 10, 2007 (published as WO2007/134161) and in U.S. patent application Ser. No. 12/478,452 filedJun. 4, 2009 (published as US 2010/0010066) and International PatentApplication No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO2009/134487). All are incorporated by reference in their entirety forall purposes.

The sequences of siRNA targeting a PCSK9 gene are described in Table 1and Table 2 above, and Tables 4-8 below.

Example 3. XBP-1 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using XBP-1 siRNA canbe found in detail in U.S. patent application Ser. No. 12/425,811 filedon Apr. 17, 2009 and published as US 2009-0275638. This application isincorporated by reference in its entirety for all purposes.

The sequences of siRNA targeting a XBP-1 gene are described in Table 3above, and Tables 9-13 below.

Example 4. A Dual Targeting siRNA Agent

A dual targeting siRNA agent was synthesized. The sense and antisensestrands for AD-10792 (target gene is PCSK9, see Table 2)) and AD-18038(target gene is XBP-1, see Table 3) were synthesized. The two sensestrands were covalently bound using a disulfide linker “Q51” with thestructure shown below.

The resulting dual sense strand was hybridized to the correspondingantisense strands to create a 42 mer dual targeting siRNA agent“AD-23426” (SEQ ID NOS 4162-4165, respectively, in order of appearance):

GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTsdTdTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAcAGA

Example 5. Inhibition of PCSK9 and Xbp-1 mRNA Levels by the PCSK9-Xbp1Dual Targeting siRNA in Primary Mouse Hepatocytes

Primary mouse hepatocytes were transfected with dual targeting AD-23426or individual siRNAs (AD-10792 and AD-18038) in lipofectamine 2000(Invitrogen protocol). 48 hours after transfection cells were harvestedand lysed. PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA incell lysates prepared according to manufacturer's protocol. PCSK9 toGAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase)and graphed.

As shown in FIG. 1, the dual targeting siRNA was at least as effectiveat inhibiting their corresponding target gene as the single siRNAs.

Example 6. Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction ofTotal Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA in Mice

The dual targeting AD-23426 was formulated in an LNP09 formulation:XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with alipid:siRNA ratio of about 10:1. The LNP09-AD-23426 was administered bytail vein injection into C₅₇B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6mg/kg. LNP09 formulated single siRNAs (AD-10792 and AD-18038) wereadministered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers andplasma were harvested 72 hours post-injection (5 animals per group).

PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA inlivers prepared according to the manufacturer's protocol. PCSK9 to GAPDHor Xbp-1 to GAPDH ratios were normalized to control (luciferase) andgraphed. The results are shown in FIG. 2.

Total cholesterol was measure in serum according to manufacturer'sinstructions using a cholesterol kit from WAKO Tex.

The results demonstrate that the dual targeting siRNAs were at least aseffective at inhibiting their corresponding target as single siRNAs invivo. The results also show that the dual targeting construct has anadditive effect compared to the single siRNAs at reducing total serumcholesterol.

Example 7: No Induction of IFN-α and TNF-α in HuPBMC

The effect of a dual targeting siRNA, AD-23426, on IFN-α and TNF-α inhuman PBMC was investigated.

Whole Blood anti-coagulated with Sodium Heparin was obtained fromhealthy donors at Research Blood Components, Inc (Boston, Mass.).Peripheral blood mononuclear cells (PBMC) were isolated by standardFicoll-Hypaque density centrifugation. Isolated PBMC were seeded at1×10⁵ cells/well in 96 well plates and cultured in RPMI 1640 GlutaMaxMedium (Invitrogen) supplemented with 10% heat-inactivated fetal bovineserum and 1% antibiotic/antimycotic (Invitrogen). siRNAs weretransfected using DOTAP Transfection Reagent (Roche Applied Science).DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes beforemixing with an equal volume of Opti-MEM containing the siRNA.siRNA/transfection reagent complexes were incubated for 15 minutes atroom temperature prior to being added to PBMC. siRNAs were transfectedat final concentrations of 266 nM, 133 nM or 67 nM using 16 μg/ml, 8μg/ml or 4 μg/ml DOTAP, respectively. The ratio of siRNA to DOTAP is16.5 μmol/μg. Transfected PBMC were incubated at 37° C., 5% CO₂ for 24hrs after which supernatants were harvested and stored at −80° C. untilanalysis. Quantitative cytokine analysis was done using commerciallyavailable Instant ELISA Kits for IFN-α (BMS216INST) and TNF-α(BMS223INST); both from Bender MedSystems (Vienna, Austria).

LNP09 and DOTAP formulated siRNAs were administered. Control siRNAs wereAD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and AD-18221. AD-10792:PCSK9 siRNA. AD-18038: XBP-1 siRNA.

The results are shown in FIG. 4. AD-23426 did not induce production ofIFN-α and TNF-α, similar to the result obtained with the single targetgene siRNAs. As expected, unmodified siRNAs (AD-5048 and AD-18889)induced production of both IFN-α and TNF-α. These results demonstratethat a dual targeting siRNA does not induce an immune response.

Example 8. Reduction of Total Serum Cholesterol with PCSK9-Xbp1 DualTargeting siRNA Humans

A human subject is treated with a pharmaceutical composition, e.g., anucleic acid-lipid particle having a dual targeting siRNA agent.

At time zero, a suitable first dose of the pharmaceutical composition issubcutaneously administered to the subject. The composition isformulated as described herein. After a period of time, the subject'scondition is evaluated, e.g., by measurement of total serum cholesterol.This measurement can be accompanied by a measurement of PCSK9 expressionin said subject, and/or the products of the successful siRNA-targetingof PCSK9 mRNA. Other relevant criteria can also be measured. The numberand strength of doses are adjusted according to the subject's needs.

After treatment, the subject's condition is compared to the conditionexisting prior to the treatment, or relative to the condition of asimilarly afflicted but untreated subject.

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the present disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

TABLE 4 Sequences of siRNA targeted to PCSK9 SEQ Antisense SEQ *TargetSense strand (5′-3′)¹ ID NO: strand (5′-3′)¹ ID NO: Duplex #  2-20AGCGACGUCGAGGCGCUCATT    1 UGAGCGCCUCGAC    2 AD-15220 GUCGCUTT 15-33CGCUCAUGGUUGCAGGCGGTT    3 CCGCCUGCAACCA    4 AD-15275 UGAGCGTT 16-34GCUCAUGGUUGCAGGCGGGTT    5 CCCGCCUGCAACC    6 AD-15301 AUGAGCTT 30-48GCGGGCGCCGCCGUUCAGUTT    7 ACUGAACGGCGGC    8 AD-15276 GCCCGCTT 31-49CGGGCGCCGCCGUUCAGUUTT    9 AACUGAACGGCGG   10 AD-15302 CGCCCGTT 32-50GGGCGCCGCCGUUCAGUUCTT   11 GAACUGAACGGCG   12 AD-15303 GCGCCCTT 40-58CCGUUCAGUUCAGGGUCUGTT   13 CAGACCCUGAACU   14 AD-15221 GAACGGTT 43-61UUCAGUUCAGGGUCUGAGCTT   15 GCUCAGACCCUGA   16 AD-15413 ACUGAATT  82-100GUGAGACUGGCUCGGGCGGTT   17 CCGCCCGAGCCAG   18 AD-15304 UCUCACTT 100-118GGCCGGGACGCGUCGUUGCTT   19 GCAACGACGCGUC   20 AD-15305 CCGGCCTT 101-119GCCGGGACGCGUCGUUGCATT   21 UGCAACGACGCGU   22 AD-15306 CCCGGCTT 102-120CCGGGACGCGUCGUUGCAGTT   23 CUGCAACGACGCG   24 AD-15307 UCCCGGTT 105-123GGACGCGUCGUUGCAGCAGTT   25 CUGCUGCAACGAC   26 AD-15277 GCGUCCTT 135-153UCCCAGCCAGGAUUCCGCGTsT   27 CGCGGAAUCCUGG   28 AD-9526 CUGGGATsT 135-153ucccAGccAGGAuuccGcGTsT   29 CGCGGAAUCCUGG   30 AD-9652 CUGGGATsT 136-154CCCAGCCAGGAUUCCGCGCTsT   31 GCGCGGAAUCCUG   32 AD-9519 GCUGGGTsT 136-154cccAGccAGGAuuccGcGcTsT   33 GCGCGGAAUCCUG   34 AD-9645 GCUGGGTsT 138-156CAGCCAGGAUUCCGCGCGCTsT   35 GCGCGCGGAAUCC   36 AD-9523 UGGCUGTsT 138-156cAGccAGGAuuccGcGcGcTsT   37 GCGCGCGGAAUCC   38 AD-9649 UGGCUGTsT 185-203AGCUCCUGCACAGUCCUCCTsT   39 GGAGGACUGUGCA   40 AD-9569 GGAGCUTsT 185-203AGcuccuGcAcAGuccuccTsT   41 GGAGGACUGUGcA   42 AD-9695 GGAGCUTsT 205-223CACCGCAAGGCUCAAGGCGTT   43 CGCCUUGAGCCUU   44 AD-15222 GCGGUGTT 208-226CGCAAGGCUCAAGGCGCCGTT   45 CGGCGCCUUGAGC   46 AD-15278 CUUGCGTT 210-228CAAGGCUCAAGGCGCCGCCTT   47 GGCGGCGCCUUGA   48 AD-15178 GCCUUGTT 232-250GUGGACCGCGCACGGCCUCTT   49 GAGGCCGUGCGCG   50 AD-15308 GUCCACTT 233-251UGGACCGCGCACGGCCUCUTT   51 AGAGGCCGUGCGC   52 AD-15223 GGUCCATT 234-252GGACCGCGCACGGCCUCUATT   53 UAGAGGCCGUGCG   54 AD-15309 CGGUCCTT 235-253GACCGCGCACGGCCUCUAGTT   55 CUAGAGGCCGUGC   56 AD-15279 GCGGUCTT 236-254ACCGCGCACGGCCUCUAGGTT   57 CCUAGAGGCCGUG   58 AD-15194 CGCGGUTT 237-255CCGCGCACGGCCUCUAGGUTT   59 ACCUAGAGGCCGU   60 AD-15310 GCGCGGTT 238-256CGCGCACGGCCUCUAGGUCTT   61 GACCUAGAGGCCG   62 AD-15311 UGCGCGTT 239-257GCGCACGGCCUCUAGGUCUTT   63 AGACCUAGAGGCC   64 AD-15392 GUGCGCTT 240-258CGCACGGCCUCUAGGUCUCTT   65 GAGACCUAGAGGC   66 AD-15312 CGUGCGTT 248-266CUCUAGGUCUCCUCGCCAGTT   67 CUGGCGAGGAGAC   68 AD-15313 CUAGAGTT 249-267UCUAGGUCUCCUCGCCAGGTT   69 CCUGGCGAGGAGA   70 AD-15280 CCUAGATT 250-268CUAGGUCUCCUCGCCAGGATT   71 UCCUGGCGAGGAG   72 AD-15267 ACCUAGTT 252-270AGGUCUCCUCGCCAGGACATT   73 UGUCCUGGCGAGG   74 AD-15314 AGACCUTT 258-276CCUCGCCAGGACAGCAACCTT   75 GGUUGCUGUCCUG   76 AD-15315 GCGAGGTT 300-318CGUCAGCUCCAGGCGGUCCTsT   77 GGACCGCCUGGAG   78 AD-9624 CUGACGTsT 300-318cGucAGcuccAGGcGGuccTsT   79 GGACCGCCUGGAG   80 AD-9750 CUGACGTsT 301-319GUCAGCUCCAGGCGGUCCUTsT   81 AGGACCGCCUGGA   82 AD-9623 GCUGACTsT 301-319GucAGcuccAGGcGGuccuTsT   83 AGGACCGCCUGGA   84 AD-9749 GCUGACTsT 370-388GGCGCCCGUGCGCAGGAGGTT   85 CCUCCUGCGCACG   86 AD-15384 GGCGCCTT 408-426GGAGCUGGUGCUAGCCUUGTsT   87 CAAGGCUAGCACC   88 AD-9607 AGCUCCTsT 408-426GGAGcuGGuGcuAGccuuGTsT   89 cAAGGCuAGcACc   90 AD-9733 AGCUCCTsT 411-429GCUGGUGCUAGCCUUGCGUTsT   91 ACGCAAGGCUAGC   92 AD-9524 ACCAGCTsT 411-429GcuGGuGcuAGccuuGcGuTsT   93 ACGcAAGGCuAGc   94 AD-9650 ACcAGCTsT 412-430CUGGUGCUAGCCUUGCGUUTsT   95 AACGCAAGGCUAG   96 AD-9520 CACCAGTsT 412-430CUGGUGCUAGCCUUGCGUUTsT   97 AACGCAAGGCUAG   98 AD-9520 CACCAGTsT 412-430cuGGuGcuAGccuuGcGuuTsT   99 AACGcAAGGCuAG  100 AD-9646 cACcAGTsT 416-434UGCUAGCCUUGCGUUCCGATsT  101 UCGGAACGCAAGG  102 AD-9608 CUAGCATsT 416-434uGcuAGccuuGcGuuccGATsT  103 UCGGAACGcAAGG  104 AD-9734 CuAGcATsT 419-437UAGCCUUGCGUUCCGAGGATsT  105 UCCUCGGAACGCA  106 AD-9546 AGGCUATsT 419-437uAGccuuGcGuuccGAGGATsT  107 UCCUCGGAACGcA  108 AD-9672 AGGCuATsT 439-457GACGGCCUGGCCGAAGCACTT  109 GUGCUUCGGCCAG  110 AD-15385 GCCGUCTT 447-465GGCCGAAGCACCCGAGCACTT  111 GUGCUCGGGUGCU  112 AD-15393 UCGGCCTT 448-466GCCGAAGCACCCGAGCACGTT  113 CGUGCUCGGGUGC  114 AD-15316 UUCGGCTT 449-467CCGAAGCACCCGAGCACGGTT  115 CCGUGCUCGGGUG  116 AD-15317 CUUCGGTT 458-476CCGAGCACGGAACCACAGCTT  117 GCUGUGGUUCCGU  118 AD-15318 GCUCGGTT 484-502CACCGCUGCGCCAAGGAUCTT  119 GAUCCUUGGCGCA  120 AD-15195 GCGGUGTT 486-504CCGCUGCGCCAAGGAUCCGTT  121 CGGAUCCUUGGCG  122 AD-15224 CAGCGGTT 487-505CGCUGCGCCAAGGAUCCGUTT  123 ACGGAUCCUUGGC  124 AD-15188 GCAGCGTT 489-507CUGCGCCAAGGAUCCGUGGTT  125 CCACGGAUCCUUG  126 AD-15225 GCGCAGTT 500-518AUCCGUGGAGGUUGCCUGGTT  127 CCAGGCAACCUCC  128 AD-15281 ACGGAUTT 509-527GGUUGCCUGGCACCUACGUTT  129 ACGUAGGUGCCAG  130 AD-15282 GCAACCTT 542-560AGGAGACCCACCUCUCGCATT  131 UGCGAGAGGUGGG  132 AD-15319 UCUCCUTT 543-561GGAGACCCACCUCUCGCAGTT  133 CUGCGAGAGGUGG  134 AD-15226 GUCUCCTT 544-562GAGACCCACCUCUCGCAGUTT  135 ACUGCGAGAGGUG  136 AD-15271 GGUCUCTT 549-567CCACCUCUCGCAGUCAGAGTT  137 CUCUGACUGCGAG  138 AD-15283 AGGUGGTT 552-570CCUCUCGCAGUCAGAGCGCTT  139 GCGCUCUGACUGC  140 AD-15284 GAGAGGTT 553-571CUCUCGCAGUCAGAGCGCATT  141 UGCGCUCUGACUG  142 AD-15189 CGAGAGTT 554-572UCUCGCAGUCAGAGCGCACTT  143 GUGCGCUCUGACU  144 AD-15227 GCGAGATT 555-573CUCGCAGUCAGAGCGCACUTsT  145 AGUGCGCUCUGAC  146 AD-9547 UGCGAGTsT 555-573cucGcAGucAGAGcGcAcuTsT  147 AGUGCGCUCUGAC  148 AD-9673 UGCGAGTsT 558-576GCAGUCAGAGCGCACUGCCTsT  149 GGCAGUGCGCUCU  150 AD-9548 GACUGCTsT 558-576GcAGucAGAGcGcAcuGccTsT  151 GGcAGUGCGCUCU  152 AD-9674 GACUGCTsT 606-624GGGAUACCUCACCAAGAUCTsT  153 GAUCUUGGUGAGG  154 AD-9529 UAUCCCTsT 606-624GGGAuAccucAccAAGAucTsT  155 GAUCUUGGUGAGG  156 AD-9655 uAUCCCTsT 659-677UGGUGAAGAUGAGUGGCGATsT  157 UCGCCACUCAUCU  158 AD-9605 UCACCATsT 659-677uGGuGAAGAuGAGuGGcGATsT  159 UCGCcACUcAUCU  160 AD-9731 UcACcATsT 663-681GAAGAUGAGUGGCGACCUGTsT  161 CAGGUCGCCACUC  162 AD-9596 AUCUUCTsT 663-681GAAGAuGAGuGGcGAccuGTsT  163 cAGGUCGCcACUc  164 AD-9722 AUCUUCTsT 704-722CCCAUGUCGACUACAUCGATsT  165 UCGAUGUAGUCGA  166 AD-9583 CAUGGGTsT 704-722cccAuGucGAcuAcAucGATsT  167 UCGAUGuAGUCGA  168 AD-9709 cAUGGGTsT 718-736AUCGAGGAGGACUCCUCUGTsT  169 CAGAGGAGUCCUC  170 AD-9579 CUCGAUTsT 718-736AucGAGGAGGAcuccucuGTsT  171 cAGAGGAGUCCUC  172 AD-9705 CUCGAUTsT 758-776GGAACCUGGAGCGGAUUACTT  173 GUAAUCCGCUCCA  174 AD-15394 GGUUCCTT 759-777GAACCUGGAGCGGAUUACCTT  175 GGUAAUCCGCUCC  176 AD-15196 AGGUUCTT 760-778AACCUGGAGCGGAUUACCCTT  177 GGGUAAUCCGCUC  178 AD-15197 CAGGUUTT 777-795CCCUCCACGGUACCGGGCGTT  179 CGCCCGGUACCGU  180 AD-15198 GGAGGGTT 782-800CACGGUACCGGGCGGAUGATsT  181 UCAUCCGCCCGGU  182 AD-9609 ACCGUGTsT 782-800cAcGGuAccGGGcGGAuGATsT  183 UcAUCCGCCCGGu  184 AD-9735 ACCGUGTsT 783-801ACGGUACCGGGCGGAUGAATsT  185 UUCAUCCGCCCGG  186 AD-9537 UACCGUTsT 783-801AcGGuAccGGGcGGAuGAATsT  187 UUcAUCCGCCCGG  188 AD-9663 uACCGUTsT 784-802CGGUACCGGGCGGAUGAAUTsT  189 AUUCAUCCGCCCG  190 AD-9528 GUACCGTsT 784-802cGGuAccGGGcGGAuGAAuTsT  191 AUUcAUCCGCCCG  192 AD-9654 GuACCGTsT 785-803GGUACCGGGCGGAUGAAUATsT  193 UAUUCAUCCGCCC  194 AD-9515 GGUACCTsT 785-803GGuAccGGGcGGAuGAAuATsT  195 uAUUcAUCCGCCC  196 AD-9641 GGuACCTsT 786-804GUACCGGGCGGAUGAAUACTsT  197 GUAUUCAUCCGCC  198 AD-9514 CGGUACTsT 786-804GuAccGGGcGGAuGAAuAcTsT  199 GuAUUcAUCCGCC  200 AD-9640 CGGuACTsT 788-806ACCGGGCGGAUGAAUACCATsT  201 UGGUAUUCAUCCG  202 AD-9530 CCCGGUTsT 788-806AccGGGcGGAuGAAuAccATsT  203 UGGuAUUcAUCCG  204 AD-9656 CCCGGUTsT 789-807CCGGGCGGAUGAAUACCAGTsT  205 CUGGUAUUCAUCC  206 AD-9538 GCCCGGTsT 789-807ccGGGcGGAuGAAuAccAGTsT  207 CUGGuAUUcAUCC  208 AD-9664 GCCCGGTsT 825-843CCUGGUGGAGGUGUAUCUCTsT  209 GAGAUACACCUCC  210 AD-9598 ACCAGGTsT 825-843ccuGGuGGAGGuGuAucucTsT  211 GAGAuAcACCUCc  212 AD-9724 ACcAGGTsT 826-844CUGGUGGAGGUGUAUCUCCTsT  213 GGAGAUACACCUC  214 AD-9625 CACCAGTsT 826-844cuGGuGGAGGuGuAucuccTsT  215 GGAGAuAcACCUC  216 AD-9751 cACcAGTsT 827-845UGGUGGAGGUGUAUCUCCUTsT  217 AGGAGAUACACCU  218 AD-9556 CCACCATsT 827-845uGGuGGAGGuGuAucuccuTsT  219 AGGAGAuAcACCU  220 AD-9682 CcACcATsT 828-846GGUGGAGGUGUAUCUCCUATsT  221 UAGGAGAUACACC  222 AD-9539 UCCACCTsT 828-846GGuGGAGGuGuAucuccuATsT  223 uAGGAGAuAcACC  224 AD-9665 UCcACCTsT 831-849GGAGGUGUAUCUCCUAGACTsT  225 GUCUAGGAGAUAC  226 AD-9517 ACCUCCTsT 831-849GGAGGuGuAucuccuAGAcTsT  227 GUCuAGGAGAuAc  228 AD-9643 ACCUCCTsT 833-851AGGUGUAUCUCCUAGACACTsT  229 GUGUCUAGGAGAU  230 AD-9610 ACACCUTsT 833-851AGGuGuAucuccuAGAcAcTsT  231 GUGUCuAGGAGAu  232 AD-9736 AcACCUTsT 833-851AfgGfuGfuAfuCfuCfcUfaG  233 P*gUfgUfcUfaG  234 AD-14681 faCfaCfTsTfgAfgAfuAfcAf cCfuTsT 833-851 AGGUfGUfAUfCfUfCfCfUfA  235 GUfGUfCfUfAGG 236 AD-14691 GACfACfTsT AGAUfACfACfCf UfTsT 833-851AgGuGuAuCuCcUaGaCaCTsT  237 P*gUfgUfcUfaG  238 AD-14701 fgAfgAfuAfcAfcCfuTsT 833-851 AgGuGuAuCuCcUaGaCaCTsT  239 GUfGUfCfUfAGG  240 AD-14711AGAUfACfACfCf UfTsT 833-851 AfgGfuGfuAfuCfuCfcUfaG  241 GUGUCuaGGagAU 242 AD-14721 faCfaCfTsT ACAccuTsT 833-851 AGGUfGUfAUfCfUfCfCfUfA  243GUGUCuaGGagAU  244 AD-14731 GACfACfTsT ACAccuTsT 833-851AgGuGuAuCuCcUaGaCaCTsT  245 GUGUCuaGGagAU  246 AD-14741 ACAccuTsT833-851 GfcAfcCfcUfcAfuAfgGfcC  247 P*uCfcAfgGfcC  248 AD-15087fuGfgAfTsT fuAfuGfaGfgGf uGfcTsT 833-851 GCfACfCfCfUfCfAUfAGGCf  249UfCfCfAGGCfCf  250 AD-15097 CfUfGGATsT UfAUfGAGGGUfG CfTsT 833-851GcAcCcUcAuAgGcCuGgATsT  251 P*uCfcAfgGfcC  252 AD-15107 fuAfuGfaGfgGfuGfcTsT 833-851 GcAcCcUcAuAgGcCuGgATsT  253 UfCfCfAGGCfCf  254 AD-15117UfAUfGAGGGUfG CfTsT 833-851 GfcAfcCfcUfcAfuAfgGfcC  255 UCCAGgcCUauGA 256 AD-15127 fuGfgAfTsT GGGugcTsT 833-851 GCfACfCfCfUfCfAUfAGGCf  257UCCAGgcCUauGA  258 AD-15137 CfUfGGATsT GGGugcTsT 833-851GcAcCcUcAuAgGcCuGgATsT  259 UCCAGgcCUauGA  260 AD-15147 GGGugcTsT836-854 UGUAUCUCCUAGACACCAGTsT  261 CUGGUGUCUAGGA  262 AD-9516 GAUACATsT836-854 uGuAucuccuAGAcAccAGTsT  263 CUGGUGUCuAGGA  264 AD-9642 GAuAcATsT840-858 UCUCCUAGACACCAGCAUATsT  265 UAUGCUGGUGUCU  266 AD-9562 AGGAGATsT840-858 ucuccuAGAcAccAGcAuATsT  267 uAUGCUGGUGUCu  268 AD-9688 AGGAGATsT840-858 UfcUfcCfuAfgAfcAfcCfaG  269 P*uAfuGfcUfgG  270 AD-14677fcAfuAfTsT fuGfuCfuAfgGf aGfaTsT 840-858 UfCfUfCfCfUfAGACfACfCf  271UfAUfGCfUfGGU  272 AD-14687 AGCfAUfATsT fGUfCfUfAGGAG ATsT 840-858UcUcCuAgAcAcCaGcAuATsT  273 P*uAfuGfcUfgG  274 AD-14697 fuGfuCfuAfgGfaGfaTsT 840-858 UcUcCuAgAcAcCaGcAuATsT  275 UfAUfGCfUfGGU  276 AD-14707fGUfCfUfAGGAG ATsT 840-858 UfcUfcCfuAafAfcAfcCfaG  277 UAUGCugGUguCU 278 AD-14717 fcAfuAfTsT AGGagaTsT 840-858 UfCfUfCfCfUfAGACfACfCf  279UAUGCugGUguCU  280 AD-14727 AGCfAUfATsT AGGagaTsT 840-858UcUcCuAgAcAcCaGcAuATsT  281 UAUGCugGUguCU  282 AD-14737 AGGagaTsT840-858 AfgGfcCfuGfgAfgUfuUfaU  283 P*cCfgAfaUfaA  284 AD-15083fuCfgGfTsT faCfuCfcAfgGf cCfuTsT 840-858 AGGCfCfUfGGAGUfUfUfAUf  285CfCfGAAUfAAAC  286 AD-15093 UfCfGGTsT fUfCfCfAGGCfC fUfTsT 840-858AgGcCuGgAgUuUaUuCgGTsT  287 P*cCfgAfaUfaA  288 AD-15103 faCfuCfcAfgGfcCfuTsT 840-858 AgGcCuGgAgUuUaUuCgGTsT  289 CfCfGAAUfAAAC  290 AD-15113fUfCfCfAGGCfC fUfTsT 840-858 AfgGfcCfuGfgAfgUfuUfaU  291 CCGAAuaAAcuCC 292 AD-15123 fuCfgGfTsT AGGccuTsT 840-858 AGGCfCfUfGGAGUfUfUfAUf  293CCGAAuaAAcuCC  294 AD-15133 UfCfGGTsT AGGccuTsT 840-858AgGcCuGgAgUuUaUuCgGTsT  295 CCGAAuaAAcuCC  296 AD-15143 AGGccuTsT841-859 CUCCUAGACACCAGCAUACTsT  297 GUAUGCUGGUGUC  298 AD-9521 UAGGAGTsT841-859 cuccuAGAcAccAGcAuAcTsT  299 GuAUGCUGGUGUC  300 AD-9647 uAGGAGTsT842-860 UCCUAGACACCAGCAUACATsT  301 UGUAUGCUGGUGU  302 AD-9611 CUAGGATsT842-860 uccuAGAcAccAGcAuAcATsT  303 UGuAUGCUGGUGU  304 AD-9737 CuAGGATsT843-861 CCUAGACACCAGCAUACAGTsT  305 CUGUAUGCUGGUG  306 AD-9592 UCUAGGTsT843-861 ccuAGAcAccAGcAuAcAGTsT  307 CUGuAUGCUGGUG  308 AD-9718 UCuAGGTsT847-865 GACACCAGCAUACAGAGUGTsT  309 CACUCUGUAUGCU  310 AD-9561 GGUGUCTsT847-865 GAcAccAGcAuAcAGAGuGTsT  311 cACUCUGuAUGCU  312 AD-9687 GGUGUCTsT855-873 CAUACAGAGUGACCACCGGTsT  313 CCGGUGGUCACUC  314 AD-9636 UGUAUGTsT855-873 cAuAcAGAGuGAccAccGGTsT  315 CCGGUGGUcACUC  316 AD-9762 UGuAUGTsT860-878 AGAGUGACCACCGGGAAAUTsT  317 AUUUCCCGGUGGU  318 AD-9540 CACUCUTsT860-878 AGAGuGAccAccGGGAAAuTsT  319 AUUUCCCGGUGGU  320 AD-9666 cACUCUTsT861-879 GAGUGACCACCGGGAAAUCTsT  321 GAUUUCCCGGUGG  322 AD-9535 UCACUCTsT861-879 GAGuGAccAccGGGAAAucTsT  323 GAUUUCCCGGUGG  324 AD-9661 UcACUCTsT863-881 GUGACCACCGGGAAAUCGATsT  325 UCGAUUUCCCGGU  326 AD-9559 GGUCACTsT863-881 GuGAccAccGGGAAAucGATsT  327 UCGAUUUCCCGGU  328 AD-9685 GGUcACTsT865-883 GACCACCGGGAAAUCGAGGTsT  329 CCUCGAUUUCCCG  330 AD-9533 GUGGUCTsT865-883 GAccAccGGGAAAucGAGGTsT  331 CCUCGAUUUCCCG  332 AD-9659 GUGGUCTsT866-884 ACCACCGGGAAAUCGAGGGTsT  333 CCCUCGAUUUCCC  334 AD-9612 GGUGGUTsT866-884 AccAccGGGAAAucGAGGGTsT  335 CCCUCGAUUUCCC  336 AD-9738 GGUGGUTsT867-885 CCACCGGGAAAUCGAGGGCTsT  337 GCCCUCGAUUUCC  338 AD-9557 CGGUGGTsT867-885 ccAccGGGAAAucGAGGGcTsT  339 GCCCUCGAUUUCC  340 AD-9683 CGGUGGTsT875-893 AAAUCGAGGGCAGGGUCAUTsT  341 AUGACCCUGCCCU  342 AD-9531 CGAUUUTsT875-893 AAAucGAGGGcAGGGucAuTsT  343 AUGACCCUGCCCU  344 AD-9657 CGAUUUTsT875-893 AfaAfuCfgAfgGfgCfaGfgG  345 P*aUfgAfcCfcU  346 AD-14673fuCfaUfTsT fgCfcCfuCfgAf uUfuTsT 875-893 AAAUfCfGAGGGCfAGGGUfCf  347AUfGACfCfCfUf  348 AD-14683 AUfTsT GCfCfCfUfCfGA UfUfUfTsT 875-893AaAuCgAgGgCaGgGuCaUTsT  349 P*aUfgAfcCfcU  350 AD-14693 fgCfcCfuCfgAfuUfuTsT 875-893 AaAuCgAgGgCaGgGuCaUTsT  351 AUfGACfCfCfUf  352 AD-14703GCfCfCfUfCfGA UfUfUfTsT 875-893 AfaAfuCfgAfgGfgCfaGfgG  353AUGACccUGccCU  354 AD-14713 fuCfaUfTsT CGAuuuTsT 875-893AAAUfCfGAGGGCfAGGGUfCf  355 AUGACccUGccCU  356 AD-14723 AUfTsT CGAuuuTsT875-893 AaAuCgAgGgCaGgGuCaUTsT  357 AUGACccUGccCU  358 AD-14733CGAuuuTsT 875-893 CfgGfcAfcCfcUfcAfuAfgG  359 P*cAfgGfcCfuA  360AD-15079 fcCfuGfTsT fuGfaGfgGfuGf cCfgTsT 875-893 CfGGCfACfCfCfUfCfAUfAG 361 CfAGGCfCfUfAU  362 AD-15089 GCfCfUfGTsT fGAGGGUfGCfCf GTsT 875-893CgGcAcCcUcAuAgGcCuGTsT  363 P*cAfgGfcCfuA  364 AD-15099 fuGfaGfgGfuGfcCfgTsT 875-893 CgGcAcCcUcAuAgGcCuGTsT  365 CfAGGCfCfUfAU  366 AD-15109fGAGGGUfGCfCf GTsT 875-893 CfgGfcAfcCfcUfcAfuAfgG  367 CAGGCcuAUgaGG 368 AD-15119 fcCfuGfTsT GUGccgTsT 875-893 CfGGCfACfCfCfUfCfAUfAG  369CAGGCcuAUgaGG  370 AD-15129 GCfCfUfGTsT GUGccgTsT 875-893CgGcAcCcUcAuAgGcCuGTsT  371 CAGGCcuAUgaGG  372 AD-15139 GUGccgTsT877-895 AUCGAGGGCAGGGUCAUGGTsT  373 CCAUGACCCUGCC  374 AD-9542 CUCGAUTsT877-895 AucGAGGGcAGGGucAuGGTsT  375 CcAUGACCCUGCC  376 AD-9668 CUCGAUTsT878-896 cGAGGGcAGGGucAuGGucTsT  377 GACcAUGACCCUG  378 AD-9739 CCCUCGTsT880-898 GAGGGCAGGGUCAUGGUCATsT  379 UGACCAUGACCCU  380 AD-9637 GCCCUCTsT880-898 GAGGGcAGGGucAuGGucATsT  381 UGACcAUGACCCU  382 AD-9763 GCCCUCTsT882-900 GGGCAGGGUCAUGGUCACCTsT  383 GGUGACCAUGACC  384 AD-9630 CUGCCCTsT882-900 GGGcAGGGucAuGGucAccTsT  385 GGUGACcAUGACC  386 AD-9756 CUGCCCTsT885-903 CAGGGUCAUGGUCACCGACTsT  387 GUCGGUGACCAUG  388 AD-9593 ACCCUGTsT885-903 cAGGGucAuGGucAccGAcTsT  389 GUCGGUGACcAUG  390 AD-9719 ACCCUGTsT886-904 AGGGUCAUGGUCACCGACUTsT  391 AGUCGGUGACCAU  392 AD-9601 GACCCUTsT886-904 AGGGucAuGGucAccGAcuTsT  393 AGUCGGUGACcAU  394 AD-9727 GACCCUTsT892-910 AUGGUCACCGACUUCGAGATsT  395 UCUCGAAGUCGGU  396 AD-9573 GACCAUTsT892-910 AuGGucAccGAcuucGAGATsT  397 UCUCGAAGUCGGU  398 AD-9699 GACcAUTsT899-917 CCGACUUCGAGAAUGUGCCTT  399 GGCACAUUCUCGA  400 AD-15228 AGUCGGTT921-939 GGAGGACGGGACCCGCUUCTT  401 GAAGCGGGUCCCG  402 AD-15395 UCCUCCTT993-1011 CAGCGGCCGGGAUGCCGGCTsT  403 GCCGGCAUCCCGG  404 AD-9602CCGCUGTsT  993-1011 cAGcGGccGGGAuGccGGcTsT  405 GCCGGcAUCCCGG  406AD-9728 CCGCUGTsT 1020-1038 GGGUGCCAGCAUGCGCAGCTT  407 GCUGCGCAUGCUG 408 AD-15386 GCACCCTT 1038-1056 CCUGCGCGUGCUCAACUGCTsT  409GCAGUUGAGCACG  410 AD-9580 CGCAGGTsT 1038-1056 ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACG  412 AD-9706 CGcAGGTsT 1040-1058UGCGCGUGCUCAACUGCCATsT  413 UGGCAGUUGAGCA  414 AD-9581 CGCGCATsT1040-1058 uGcGcGuGcucAAcuGccATsT  415 UGGcAGUUGAGcA  416 AD-9707CGCGcATsT 1042-1060 CGCGUGCUCAACUGCCAAGTsT  417 CUUGGCAGUUGAG  418AD-9543 CACGCGTsT 1042-1060 cGcGuGcucAAcuGccAAGTsT   419 CUUGGcAGUUGAG 420 AD-9669 cACGCGTsT 1053-1071 CUGCCAAGGGAAGGGCACGTsT  421CGUGCCCUUCCCU  422 AD-9574 UGGCAGTsT 1053-1071 cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCU  424 AD-9700 UGGcAGTsT 1057-1075CAAGGGAAGGGCACGGUUATT  425 UAACCGUGCCCUU  426 AD-15320 CCCUUGTT1058-1076 AAGGGAAGGGCACGGUUAGTT  427 CUAACCGUGCCCU  428 AD-15321UCCCUUTT 1059-1077 AGGGAAGGGCACGGUUAGCTT  429 GCUAACCGUGCCC  430AD-15199 UUCCCUTT 1060-1078 GGGAAGGGCACGGUUAGCGTT  431 CGCUAACCGUGCC 432 AD-15167 CUUCCCTT 1061-1079 GGAAGGGCACGGUUAGCGGTT  433CCGCUAACCGUGC  434 AD-15164 CCUUCCTT 1062-1080 GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUG  436 AD-15166 CCCUUCTT 1063-1081AAGGGCACGGUUAGCGGCATT  437 UGCCGCUAACCGU  438 AD-15322 GCCCUUTT1064-1082 AGGGCACGGUUAGCGGCACTT  439 GUGCCGCUAACCG  440 AD-15200UGCCCUTT 1068-1086 CACGGUUAGCGGCACCCUCTT  441 GAGGGUGCCGCUA  442AD-15213 ACCGUGTT 1069-1087 ACGGUUAGCGGCACCCUCATT  443 UGAGGGUGCCGCU 444 AD-15229 AACCGUTT 1072-1090 GUUAGCGGCACCCUCAUAGTT  445CUAUGAGGGUGCC  446 AD-15215 GCUAACTT 1073-1091 UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGC  448 AD-15214 CGCUAATT 1076-1094GCGGCACCCUCAUAGGCCUTsT  449 AGGCCUAUGAGGG  450 AD-9315 UGCCGCTsT1079-1097 GCACCCUCAUAGGCCUGGATsT  451 UCCAGGCCUAUGA  452 AD-9326GGGUGCTsT 1085-1103 UCAUAGGCCUGGAGUUUAUTsT  453 AUAAACUCCAGGC  454AD-9318 CUAUGATsT 1090-1108 GGCCUGGAGUUUAUUCGGATsT  455 UCCGAAUAAACUC 456 AD-9323 CAGGCCTsT 1091-1109 GCCUGGAGUUUAUUCGGAATsT  457UUCCGAAUAAACU  458 AD-9314 CCAGGCTsT 1091-1109 GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACU  460 AD-10792 CcAGGCTsT 1091-1109GccuGGAGuuuAuucGGAATsT  461 UUCCGAAUAACUC  462 AD-10796 CAGGCTsT1093-1111 CUGGAGUUUAUUCGGAAAATsT  463 UUUUCCGAAUAAA  464 AD-9638CUCCAGTsT 1093-1111 cuGGAGuuuAuucGGAAAATsT  465 UUUUCCGAAuAAA  466AD-9764 CUCcAGTsT 1095-1113 GGAGUUUAUUCGGAAAAGCTsT  467 GCUUUUCCGAAUA 468 AD-9525 AACUCCTsT 1095-1113 GGAGuuuAuucGGAAAAGcTsT  469GCUUUUCCGAAuA  470 AD-9651 AACUCCTsT 1096-1114 GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAU  472 AD-9560 AAACUCTsT 1096-1114GAGuuuAuucGGAAAAGccTsT  473 GGCUUUUCCGAAu  474 AD-9686 AAACUCTsT1100-1118 UUAUUCGGAAAAGCCAGCUTsT  475 AGCUGGCUUUUCC  476 AD-9536GAAUAATsT 1100-1118 uuAuucGGAAAAGccAGcuTsT  477 AGCUGGCUUUUCC  478AD-9662 GAAuAATsT 1154-1172 CCCUGGCGGGUGGGUACAGTsT  479 CUGUACCCACCCG 480 AD-9584 CCAGGGTsT 1154-1172 cccuGGcGGGuGGGuAcAGTsT  481CUGuACCcACCCG  482 AD-9710 CcAGGGTsT 1155-1173 CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCC  484 AD-15323 GCCAGGTT 1157-1175UGGCGGGUGGGUACAGCCGTsT  485 CGGCUGUACCCAC  486 AD-9551 CCGCCATsT1157-1175 uGGcGGGuGGGuAcAGccGTsT  487 CGGCUGuACCcAC  488 AD-9677CCGCcATsT 1158-1176 GGCGGGUGGGUACAGCCGCTT  489 GCGGCUGUACCCA  490AD-15230 CCCGCCTT 1162-1180 GGUGGGUACAGCCGCGUCCTT  491 GGACGCGGCUGUA 492 AD-15231 CCCACCTT 1164-1182 UGGGUACAGCCGCGUCCUCTT  493GAGGACGCGGCUG  494 AD-15285 UACCCATT 1172-1190 GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGA  496 AD-15396 CGCGGCTT 1173-1191CCGCGUCCUCAACGCCGCCTT  497 GGCGGCGUUGAGG  498 AD-15397 ACGCGGTT1216-1234 GUCGUGCUGGUCACCGCUGTsT  499 CAGCGGUGACCAG  500 AD-9600CACGACTsT 1216-1234 GucGuGcuGGucAccGcuGTsT  501 cAGCGGUGACcAG  502AD-9726 cACGACTsT 1217-1235 UCGUGCUGGUCACCGCUGCTsT  503 GCAGCGGUGACCA 504 AD-9606 GCACGATsT 1217-1235 ucGuGcuGGucAccGcuGcTsT  505GcAGCGGUGACcA  506 AD-9732 GcACGATsT 1223-1241 UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGG  508 AD-9633 UGACCATsT 1223-1241uGGucAccGcuGccGGcAATsT  509 UUGCCGGcAGCGG  510 AD-9759 UGACcATsT1224-1242 GGUCACCGCUGCCGGCAACTsT  511 GUUGCCGGCAGCG  512 AD-9588GUGACCTsT 1224-1242 GGucAccGcuGccGGcAAcTsT  513 GUUGCCGGcAGCG  514AD-9714 GUGACCTsT 1227-1245 CACCGCUGCCGGCAACUUCTsT  515 GAAGUUGCCGGCA 516 AD-9589 GCGGUGTsT 1227-1245 cAccGcuGccGGcAAcuucTsT  517GAAGUUGCCGGcA  518 AD-9715 GCGGUGTsT 1229-1247 CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGG  520 AD-9575 CAGCGGTsT 1229-1247ccGcuGccGGcAAcuuccGTsT  521 CGGAAGUUGCCGG  522 AD-9701 cAGCGGTsT1230-1248 CGCUGCCGGCAACUUCCGGTsT  523 CCGGAAGUUGCCG  524 AD-9563GCAGCGTsT 1230-1248 cGcuGccGGcAAcuuccGGTsT  525 CCGGAAGUUGCCG  526AD-9689 GcAGCGTsT 1231-1249 GCUGCCGGCAACUUCCGGGTsT  527 CCCGGAAGUUGCC 528 AD-9594 GGCAGCTsT 1231-1249 GcuGccGGcAAcuuccGGGTsT  529CCCGGAAGUUGCC  530 AD-9720 GGcAGCTsT 1236-1254 CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAG  532 AD-9585 UUGCCGTsT 1236-1254cGGcAAcuuccGGGAcGAuTsT  533 AUCGUCCCGGAAG  534 AD-9711 UUGCCGTsT1237-1255 GGCAACUUCCGGGACGAUGTsT  535 CAUCGUCCCGGAA  536 AD-9614GUUGCCTsT 1237-1255 GGcAAcuuccGGGAcGAuGTsT  537 cAUCGUCCCGGAA  538AD-9740 GUUGCCTsT 1243-1261 UUCCGGGACGAUGCCUGCCTsT  539 GGCAGGCAUCGUC 540 AD-9615 CCGGAATsT 1243-1261 uuccGGGAcGAuGccuGccTsT  541GGcAGGcAUCGUC  542 AD-9741 CCGGAATsT 1248-1266 GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCA  544 AD-9534 UCGUCCTsT 1248-1266GGACGAUGCCUGCCUCUACTsT  545 GUAGAGGCAGGCA  546 AD-9534 UCGUCCTsT1248-1266 GGAcGAuGccuGccucuAcTsT  547 GuAGAGGcAGGcA  548 AD-9660UCGUCCTsT 1279-1297 GCUCCCGAGGUCAUCACAGTT  549 CUGUGAUGACCUC  550AD-15324 GGGAGCTT 1280-1298 CUCCCGAGGUCAUCACAGUTT  551 ACUGUGAUGACCU 552 AD-15232 CGGGAGTT 1281-1299 UCCCGAGGUCAUCACAGUUTT  553AACUGUGAUGACC  554 AD-15233 UCGGGATT 1314-1332 CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGG  556 AD-15234 UCUUGGTT 1315-1333CAAGACCAGCCGGUGACCCTT  557 GGGUCACCGGCUG  558 AD-15286 GUCUUGTT1348-1366 ACCAACUUUGGCCGCUGUGTsT  559 CACAGCGGCCAAA  560 AD-9590GUUGGUTsT 1348-1366 AccAAcuuuGGccGcuGuGTsT  561 cAcAGCGGCcAAA  562AD-9716 GUUGGUTsT 1350-1368 CAACUUUGGCCGCUGUGUGTsT  563 CACACAGCGGCCA 564 AD-9632 AAGUUGTsT 1350-1368 cAAcuuuGGccGcuGuGuGTsT  565cAcAcAGCGGCcA  566 AD-9758 AAGUUGTsT 1360-1378 CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCAC  568 AD-9567 ACAGCGTsT 1360-1378cGcuGuGuGGAccucuuuGTsT   569 cAAAGAGGUCcAc  570 AD-9693 AcAGCGTsT1390-1408 GACAUCAUUGGUGCCUCCATsT  571 UGGAGGCACCAAU  572 AD-9586GAUGUCTsT 1390-1408 GAcAucAuuGGuGccuccATsT  573 UGGAGGcACcAAU  574AD-9712 GAUGUCTsT 1394-1412 UCAUUGGUGCCUCCAGCGATsT  575 UCGCUGGAGGCAC 576 AD-9564 CAAUGATsT 1394-1412 ucAuuGGuGccuccAGcGATsT  577UCGCUGGAGGcAC  578 AD-9690 cAAUGATsT 1417-1435 AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCA  580 AD-9616 GGUGCUTsT 1417-1435AGcAccuGcuuuGuGucAcTsT  581 GUGAcAcAAAGcA  582 AD-9742 GGUGCUTsT1433-1451 CACAGAGUGGGACAUCACATT  583 UGUGAUGUCCCAC  584 AD-15398UCUGUGTT 1486-1504 AUGCUGUCUGCCGAGCCGGTsT  585 CCGGCUCGGCAGA  586AD-9617 CAGCAUTsT 1486-1504 AuGcuGucuGccGAGccGGTsT  587 CCGGCUCGGcAGA 588 AD-9743 cAGcAUTsT 1491-1509 GUCUGCCGAGCCGGAGCUCTsT  589GAGCUCCGGCUCG  590 AD-9635 GCAGACTsT 1491-1509 GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCG  592 AD-9761 GcAGACTsT 1521-1539GUUGAGGCAGAGACUGAUCTsT  593 GAUCAGUCUCUGC  594 AD-9568 CUCAACTsT1521-1539 GuuGAGGcAGAGAcuGAucTsT  595 GAUcAGUCUCUGC  596 AD-9694CUcAACTsT 1527-1545 GCAGAGACUGAUCCACUUCTsT  597 GAAGUGGAUCAGU  598AD-9576 CUCUGCTsT 1527-1545 GcAGAGAcuGAuccAcuucTsT  599 GAAGUGGAUcAGU 600 AD-9702 CUCUGCTsT 1529-1547 AGAGACUGAUCCACUUCUCTsT  601GAGAAGUGGAUCA  602 AD-9627 GUCUCUTsT 1529-1547 AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcA  604 AD-9753 GUCUCUTsT 1543-1561UUCUCUGCCAAAGAUGUCATsT  605 UGACAUCUUUGGC  606 AD-9628 AGAGAATsT1543-1561 uucucuGccAAAGAuGucATsT  607 UGAcAUCUUUGGc  608 AD-9754AGAGAATsT 1545-1563 CUCUGCCAAAGAUGUCAUCTsT  609 GAUGACAUCUUUG  610AD-9631 GCAGAGTsT 1545-1563 cucuGccAAAGAuGucAucTsT  611 GAUGAcAUCUUUG 612 AD-9757 GcAGAGTsT 1580-1598 CUGAGGACCAGCGGGUACUTsT  613AGUACCCGCUGGU  614 AD-9595 CCUCAGTsT 1580-1598 cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGU  616 AD-9721 CCUcAGTsT 1581-1599UGAGGACCAGCGGGUACUGTsT  617 CAGUACCCGCUGG  618 AD-9544 UCCUCATsT1581-1599 uGAGGAccAGcGGGuAcuGTsT  619 cAGuACCCGCUGG  620 AD-9670UCCUcATsT 1666-1684 ACUGUAUGGUCAGCACACUTT  621 AGUGUGCUGACCA  622AD-15235 UACAGUTT 1668-1686 UGUAUGGUCAGCACACUCGTT  623 CGAGUGUGCUGAC 624 AD-15236 CAUACATT 1669-1687 GUAUGGUCAGCACACUCGGTT  625CCGAGUGUGCUGA  626 AD-15168 CCAUACTT 1697-1715 GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGG  628 AD-15174 CCAUCCTT 1698-1716GAUGGCCACAGCCGUCGCCTT  629 GGCGACGGCUGUG  630 AD-15325 GCCAUCTT1806-1824 CAAGCUGGUCUGCCGGGCCTT  631 GGCCCGGCAGACC  632 AD-15326AGCUUGTT 1815-1833 CUGCCGGGCCCACAACGCUTsT  633 AGCGUUGUGGGCC  634AD-9570 CGGCAGTsT 1815-1833 cuGccGGGcccAcAAcGcuTsT  635 AGCGUUGUGGGCC 636 AD-9696 CGGcAGTsT 1816-1834 UGCCGGGCCCACAACGCUUTsT  637AAGCGUUGUGGGC  638 AD-9566 CCGGCATsT 1816-1834 uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGC  640 AD-9692 CCGGcATsT 1818-1836CCGGGCCCACAACGCUUUUTsT  641 AAAAGCGUUGUGG  642 AD-9532 GCCCGGTsT1818-1836 ccGGGcccAcAAcGcuuuuTsT  643 AAAAGCGUUGUGG  644 AD-9658GCCCGGTsT 1820-1838 GGGCCCACAACGCUUUUGGTsT  645 CCAAAAGCGUUGU  646AD-9549 GGGCCCTsT 1820-1838 GGGcccAcAAcGcuuuuGGTsT  647 CcAAAAGCGUUGU 648 AD-9675 GGGCCCTsT 1840-1858 GGUGAGGGUGUCUACGCCATsT  649UGGCGUAGACACC  650 AD-9541 CUCACCTsT 1840-1858 GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACC  652 AD-9667 CUcACCTsT 1843-1861GAGGGUGUCUACGCCAUUGTsT  653 CAAUGGCGUAGAC  654 AD-9550 ACCCUCTsT1843-1861 GAGGGuGucuAcGccAuuGTsT  655 cAAUGGCGuAGAc  656 AD-9676ACCCUCTsT 1861-1879 GCCAGGUGCUGCCUGCUACTsT  657 GUAGCAGGCAGCA  658AD-9571 CCUGGCTsT 1861-1879 GccAGGuGcuGccuGcuAcTsT  659 GuAGcAGGcAGcA 660 AD-9697 CCUGGCTsT 1862-1880 CCAGGUGCUGCCUGCUACCTsT  661GGUAGCAGGCAGC  662 AD-9572 ACCUGGTsT 1862-1880 ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGc  664 AD-9698 ACCUGGTsT 2008-2026ACCCACAAGCCGCCUGUGCTT  665 GCACAGGCGGCUU  666 AD-15327 GUGGGUTT2023-2041 GUGCUGAGGCCACGAGGUCTsT  667 GACCUCGUGGCCU  668 AD-9639CAGCACTsT 2023-2041 GuGcuGAGGccAcGAGGucTsT  669 GACCUCGUGGCCU  670AD-9765 cAGcACTsT 2024-2042 UGCUGAGGCCACGAGGUCATsT  671 UGACCUCGUGGCC 672 AD-9518 UCAGCATsT 2024-2042 UGCUGAGGCCACGAGGUCATsT  673UGACCUCGUGGCC  674 AD-9518 UCAGCATsT 2024-2042 uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCC  676 AD-9644 UcAGcATsT 2024-2042UfgCfuGfaGfgCfcAfcGfaG  677 P*uGfaCfcUfcG  678 AD-14672 fgUfcAfTsTfuGfgCfcUfcAf gCfaTsT 2024-2042 UfGCfUfGAGGCfCfACfGAGG  679UfGACfCfUfCfG  680 AD-14682 UfCfATsT UfGGCfCfUfCfA GCfATsT 2024-2042UgCuGaGgCcAcGaGgUcATsT  681 P*uGfaCfcUfcG  682 AD-14692 fuGfgCfcUfcAfgCfaTsT 2024-2042 UgCuGaGgCcAcGaGgUcATsT  683 UfGACfCfUfCfG  684AD-14702 UfGGCfCfUfCfA GCfATsT 2024-2042 UfgCfuGfaGfgCfcAfcGfaG  685UGACCucGUggCC  686 AD-14712 fgUfcAfTsT UCAgcaTsT 2024-2042UfGCfUfGAGGCfCfACfGAGG  687 UGACCucGUggCC  688 AD-14722 UfCfATsTUCAgcaTsT 2024-2042 UgCuGaGgCcAcGaGgUcATsT  689 UGACCucGUggCC  690AD-14732 UCAgcaTsT 2024-2042 GfuGfgUfcAfgCfgGfcCfgG  691 P*cAfuCfcCfgG 692 AD-15078 fgAfuGfTsT fcCfgCfuGfaCf cAfcTsT 2024-2042GUfGGUfCfAGCfGGCfCfGGG  693 CfAUfCfCfCfGG  694 AD-15088 AUfGTsTCfCfGCfUfGACf CfACfTsT 2024-2042 GuGgUcAgCgGcCgGgAuGTsT  695P*cAfuCfcCfgG  696 AD-15098 fcCfgCfuGfaCf cAfcTsT 2024-2042GuGgUcAgCgGcCgGgAuGTsT  697 CfAUfCfCfCfGG  698 AD-15108 CfCfGCfUfGACfCfACfTsT 2024-2042 GfuGfgUfcAfgCfgGfcCfgG  699 CAUCCcgGCcgCU  700AD-15118 fgAfuGfTsT GACcacTsT 2024-2042 GUfGGUfCfAGCfGGCfCfGGG  701CAUCCcgGCcgCU  702 AD-15128 AUfGTsT GACcacTsT 2024-2042GuGgUcAgCgGcCgGgAuGTsT  703 CAUCCcgGCcgCU  704 AD-15138 GACcacTsT2030-2048 GGCCACGAGGUCAGCCCAATT  705 UUGGGCUGACCUC  706 AD-15237GUGGCCTT 2035-2053 CGAGGUCAGCCCAACCAGUTT  707 ACUGGUUGGGCUG  708AD-15287 ACCUCGTT 2039-2057 GUCAGCCCAACCAGUGCGUTT  709 ACGCACUGGUUGG 710 AD-15238 GCUGACTT 2041-2059 CAGCCCAACCAGUGCGUGGTT  711CCACGCACUGGUU  712 AD-15328 GGGCUGTT 2062-2080 CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUC  714 AD-15399 CCUGUGTT 2072-2090CCAGCAUCCACGCUUCCUGTsT  715 CAGGAAGCGUGGA  716 AD-9582 UGCUGGTsT2072-2090 ccAGcAuccAcGcuuccuGTsT  717 cAGGAAGCGUGGA  718 AD-9708UGCUGGTsT 2118-2136 AGUCAAGGAGCAUGGAAUCTsT  719 GAUUCCAUGCUCC  720AD-9545 UUGACUTsT 2118-2136 AGucAAGGAGcAuGGAAucTsT  721 GAUUCcAUGCUCC 722 AD-9671 UUGACUTsT 2118-2136 AfgUfcAfaGfgAfgCfaUfgG  723P*gAfuUfcCfaU  724 AD-14674 faAfuCfTsT fgCfuCfcUfuGf aCfuTsT 2118-2136AGUfCfAAGGAGCfAUfGGAAU  725 GAUfUfCfCfAUf  726 AD-14684 fCfTsTGCfUfCfCfUfUf GACfUfTsT 2118-2136 AgUcAaGgAgCaUgGaAuCTsT  727P*gAfuUfcCfaU  728 AD-14694 fgCfuCfcUfuGf aCfuTsT 2118-2136AgUcAaGgAgCaUgGaAuCTsT  729 GAUfUfCfCfAUf  730 AD-14704 GCfUfCfCfUfUfGACfUfTsT 2118-2136 AfgUfcAfaGfgAfgCfaUfgG  731 GAUUCcaUGcuCC  732AD-14714 faAfuCfTsT UUGacuTsT 2118-2136 AGUfCfAAGGAGCfAUfGGAAU  733GAUUCcaUGcuCC  734 AD-14724 fCfTsT UUGacuTsT 2118-2136AgUcAaGgAgCaUgGaAuCTsT  735 GAUUCcaUGcuCC  736 AD-14734 UUGacuTsT2118-2136 GfcGfgCfaCfcCfuCfaUfaG  737 P*aGfgCfcUfaU  738 AD-15080fgCfcUfTsT fgAfgGfgUfgCf cGfcTsT 2118-2136 GCfGGCfACfCfCfUfCfAUfA  739AGGCfCfUfAUfG  740 AD-15090 GGCfCfUfTsT AGGGUfGCfCfGC fTsT 2118-2136GcGgCaCcCuCaUaGgCcUTsT  741 P*aGfgCfcUfaU  742 AD-15100 fgAfgGfgUfgCfcGfcTsT 2118-2136 GcGgCaCcCuCaUaGgCcUTsT  743 AGGCfCfUfAUfG  744AD-15110 AGGGUfGCfCfGC fTsT 2118-2136 GfcGfgCfaCfcCfuCfaUfaG  745AGGCCuaUGagGG  746 AD-15120 fgCfcUfTsT UGCcgcTsT 2118-2136GCfGGCfACfCfCfUfCfAUfA  747 AGGCCuaUGagGG  748 AD-15130 GGCfCfUfTsTUGCcgcTsT 2118-2136 GcGgCaCcCuCaUaGgCcUTsT  749 AGGCCuaUGagGG  750AD-15140 UGCcgcTsT 2122-2140 AAGGAGCAUGGAAUCCCGGTsT  751 CCGGGAUUCCAUG 752 AD-9522 CUCCUUTsT 2122-2140 AAGGAGcAuGGAAucccGGTsT  753CCGGGAUUCcAUG  754 AD-9648 CUCCUUTsT 2123-2141 AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAU  756 AD-9552 GCUCCUTsT 2123-2141AGGAGcAuGGAAucccGGcTsT  757 GCCGGGAUUCcAU  758 AD-9678 GCUCCUTsT2125-2143 GAGCAUGGAAUCCCGGCCCTsT  759 GGGCCGGGAUUCC  760 AD-9618AUGCUCTsT 2125-2143 GAGcAuGGAAucccGGcccTsT  761 GGGCCGGGAUUCc  762AD-9744 AUGCUCTsT 2230-2248 GCCUACGCCGUAGACAACATT  763 UGUUGUCUACGGC 764 AD-15239 GUAGGCTT 2231-2249 CCUACGCCGUAGACAACACTT  765GUGUUGUCUACGG  766 AD-15212 CGUAGGTT 2232-2250 CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACG  768 AD-15240 GCGUAGTT 2233-2251UACGCCGUAGACAACACGUTT  769 ACGUGUUGUCUAC  770 AD-15177 GGCGUATT2235-2253 CGCCGUAGACAACACGUGUTT  771 ACACGUGUUGUCU  772 AD-15179ACGGCGTT 2236-2254 GCCGUAGACAACACGUGUGTT  773 CACACGUGUUGUC  774AD-15180 UACGGCTT 2237-2255 CCGUAGACAACACGUGUGUTT  775 ACACACGUGUUGU 776 AD-15241 CUACGGTT 2238-2256 CGUAGACAACACGUGUGUATT  777UACACACGUGUUG  778 AD-15268 UCUACGTT 2240-2258 UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGU  780 AD-15242 UGUCUATT 2241-2259AGACAACACGUGUGUAGUCTT  781 GACUACACACGUG  782 AD-15216 UUGUCUTT2242-2260 GACAACACGUGUGUAGUCATT  783 UGACUACACACGU  784 AD-15176GUUGUCTT 2243-2261 ACAACACGUGUGUAGUCAGTT  785 CUGACUACACACG  786AD-15181 UGUUGUTT 2244-2262 CAACACGUGUGUAGUCAGGTT  787 CCUGACUACACAC 788 AD-15243 GUGUUGTT 2247-2265 CACGUGUGUAGUCAGGAGCTT  789GCUCCUGACUACA  790 AD-15182 CACGUGTT 2248-2266 ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUAC  792 AD-15244 ACACGUTT 2249-2267CGUGUGUAGUCAGGAGCCGTT  793 CGGCUCCUGACUA  794 AD-15387 CACACGTT2251-2269 UGUGUAGUCAGGAGCCGGGTT  795 CCCGGCUCCUGAC  796 AD-15245UACACATT 2257-2275 GUCAGGAGCCGGGACGUCATsT  797 UGACGUCCCGGCU  798AD-9555 CCUGACTsT 2257-2275 GucAGGAGccGGGAcGucATsT  799 UGACGUCCCGGCU 800 AD-9681 CCUGACTsT 2258-2276 UCAGGAGCCGGGACGUCAGTsT  801CUGACGUCCCGGC  802 AD-9619 UCCUGATsT 2258-2276 ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGC  804 AD-9745 UCCUGATsT 2259-2277CAGGAGCCGGGACGUCAGCTsT  805 GCUGACGUCCCGG  806 AD-9620 CUCCUGTsT2259-2277 cAGGAGccGGGAcGucAGcTsT  807 GCUGACGUCCCGG  808 AD-9746CUCCUGTsT 2263-2281 AGCCGGGACGUCAGCACUATT  809 UAGUGCUGACGUC  810AD-15288 CCGGCUTT 2265-2283 CCGGGACGUCAGCACUACATT  811 UGUAGUGCUGACG 812 AD-15246 UCCCGGTT 2303-2321 CCGUGACAGCCGUUGCCAUTT  813AUGGCAACGGCUG  814 AD-15289 UCACGGTT 2317-2335 GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCA  816 AD-9324 GAUGGCTsT 2375-2393CCCAUCCCAGGAUGGGUGUTT  817 ACACCCAUCCUGG  818 AD-15329 GAUGGGTT2377-2395 CAUCCCAGGAUGGGUGUCUTT  819 AGACACCCAUCCU  820 AD-15330GGGAUGTT 2420-2438 AGCUUMAAAUGGUUCCGATT  821 UCGGAACCAUUUU  822 AD-15169AAAGCUTT 2421-2439 GCUUUAAAAUGGUUCCGACTT  823 GUCGGAACCAUUU  824AD-15201 UAAAGCTT 2422-2440 CUUMAAAUGGUUCCGACUTT  825 AGUCGGAACCAUU  826AD-15331 UUAAAGTT 2423-2441 UUUAAAAUGGUUCCGACUUTT  827 AAGUCGGAACCAU 828 AD-15190 UUUAAATT 2424-2442 UUAAAAUGGUUCCGACUUGTT  829CAAGUCGGAACCA  830 AD-15247 UUUUAATT 2425-2443 UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACC  832 AD-15248 AUUUUATT 2426-2444AAAAUGGUUCCGACUUGUCTT  833 GACAAGUCGGAAC  834 AD-15175 CAUUUUTT2427-2445 AAAUGGUUCCGACUUGUCCTT  835 GGACAAGUCGGAA  836 AD-15249CCAUUUTT 2428-2446 AAUGGUUCCGACUUGUCCCTT  837 GGGACAAGUCGGA  838AD-15250 ACCAUUTT 2431-2449 GGUUCCGACUUGUCCCUCUTT  839 AGAGGGACAAGUC 840 AD-15400 GGAACCTT 2457-2475 CUCCAUGGCCUGGCACGAGTT  841CUCGUGCCAGGCC  842 AD-15332 AUGGAGTT 2459-2477 CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGG  844 AD-15388 CCAUGGTT 2545-2563GAACUCACUCACUCUGGGUTT  845 ACCCAGAGUGAGU  846 AD-15333 GAGUUCTT2549-2567 UCACUCACUCUGGGUGCCUTT  847 AGGCACCCAGAGU  848 AD-15334GAGUGATT 2616-2634 UUUCACCAUUCAAACAGGUTT  849 ACCUGUUUGAAUG  850AD-15335 GUGAAATT 2622-2640 CAUUCAAACAGGUCGAGCUTT  851 AGCUCGACCUGUU 852 AD-15183 UGAAUGTT 2623-2641 AUUCAAACAGGUCGAGCUGTT  853CAGCUCGACCUGU  854 AD-15202 UUGAAUTT 2624-2642 UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUG  856 AD-15203 UUUGAATT 2625-2643UCAAACAGGUCGAGCUGUGTT  857 CACAGCUCGACCU  858 AD-15272 GUUUGATT2626-2644 CAAACAGGUCGAGCUGUGCTT  859 GCACAGCUCGACC  860 AD-15217UGUUUGTT 2627-2645 AAACAGGUCGAGCUGUGCUTT  861 AGCACAGCUCGAC  862AD-15290 CUGUUUTT 2628-2646 AACAGGUCGAGCUGUGCUCTT  863 GAGCACAGCUCGA 864 AD-15218 CCUGUUTT 2630-2648 CAGGUCGAGCUGUGCUCGGTT  865CCGAGCACAGCUC  866 AD-15389 GACCUGTT 2631-2649 AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCU  868 AD-15336 CGACCUTT 2633-2651GUCGAGCUGUGCUCGGGUGTT  869 CACCCGAGCACAG  870 AD-15337 CUCGACTT2634-2652 UCGAGCUGUGCUCGGGUGCTT  871 GCACCCGAGCACA  872 AD-15191GCUCGATT 2657-2675 AGCUGCUCCCAAUGUGCCGTT  873 CGGCACAUUGGGA  874AD-15390 GCAGCUTT 2658-2676 GCUGCUCCCAAUGUGCCGATT  875 UCGGCACAUUGGG 876 AD-15338 AGCAGCTT 2660-2678 UGCUCCCAAUGUGCCGAUGTT  877CAUCGGCACAUUG  878 AD-15204 GGAGCATT 2663-2681 UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACA  880 AD-15251 UUGGGATT 2665-2683CCAAUGUGCCGAUGUCCGUTT  881 ACGGACAUCGGCA  882 AD-15205 CAUUGGTT2666-2684 CAAUGUGCCGAUGUCCGUGTT  883 CACGGACAUCGGC  884 AD-15171ACAUUGTT 2667-2685 AAUGUGCCGAUGUCCGUGGTT  885 CCACGGACAUCGG  886AD-15252 CACAUUTT 2673-2691 CCGAUGUCCGUGGGCAGAATT  887 UUCUGCCCACGGA 888 AD-15339 CAUCGGTT 2675-2693 GAUGUCCGUGGGCAGAAUGTT  889CAUUCUGCCCACG  890 AD-15253 GACAUCTT 2678-2696 GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCC  892 AD-15340 ACGGACTT 2679-2697UCCGUGGGCAGAAUGACUUTT  893 AAGUCAUUCUGCC  894 AD-15291 CACGGATT2683-2701 UGGGCAGAAUGACUUDUAUTT  895 AUAAAAGUCAUUC  896 AD-15341UGCCCATT 2694-2712 ACUUUUAUUGAGCUCUUGUTT  897 ACAAGAGCUCAAU  898AD-15401 AAAAGUTT 2700-2718 AUUGAGCUCUUGUUCCGUGTT  899 CACGGAACAAGAG 900 AD-15342 CUCAAUTT 2704-2722 AGCUCUUGUUCCGUGCCAGTT  901CUGGCACGGAACA  902 AD-15343 AGAGCUTT 2705-2723 GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAAC  904 AD-15292 AAGAGCTT 2710-2728UGUUCCGUGCCAGGCAUUCTT  905 GAAUGCCUGGCAC  906 AD-15344 GGAACATT2711-2729 GUUCCGUGCCAGGCAUUCATT  907 UGAAUGCCUGGCA  908 AD-15254CGGAACTT 2712-2730 UUCCGUGCCAGGCAUUCAATT  909 UUGAAUGCCUGGC  910AD-15345 ACGGAATT 2715-2733 CGUGCCAGGCAUUCAAUCCTT  911 GGAUUGAAUGCCU 912 AD-15206 GGCACGTT 2716-2734 GUGCCAGGCAUUCAAUCCUTT  913AGGAUUGAAUGCC  914 AD-15346 UGGCACTT 2728-2746 CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGA  916 AD-15347 GGAUUGTT 2743-2761CACCAAGGAGGCAGGAUUCTsT  917 GAAUCCUGCCUCC  918 AD-9577 UUGGUGTsT2743-2761 cAccAAGGAGGcAGGAuucTsT  919 GAAUCCUGCCUCC  920 AD-9703UUGGUGTsT 2743-2761 CfaCfcAfaGfgAfgGfcAfgG  921 P*gAfaUfcCfuG  922AD-14678 faUfuCfTsT fcCfuCfcUfuGf gUfgTsT 2743-2761CfACfCfAAGGAGGCfAGGAUf  923 GAAUfCfCfUfGC  924 AD-14688 UfCfTsTfCfUfCfCfUfUf GGUfGTsT 2743-2761 CaCcAaGgAgGcAgGaUuCTsT 925P*gAfaUfcCfuG  926 AD-14698 fcCfuCfcUfuGf gUfgTsT 2743-2761CaCcAaGgAgGcAgGaUuCTsT  927 GAAUfCfCfUfGC  928 AD-14708 fCfUfCfCfUfUfGGUfGTsT 2743-2761 CfaCfcAfaGfgAfgGfcAfgG  929 GAAUCcuGCcuCC  930AD-14718 faUfuCfTsT UUGgugTsT 2743-2761 CfACfCfAAGGAGGCfAGGAUf  931GAAUCcuGCcuCC  932 AD-14728 UfCfTsT UUGgugTsT 2743-2761CaCcAaGgAgGcAgGaUuCTsT  933 GAAUCcuGCcuCC  934 AD-14738 UUGgugTsT2743-2761 GfgCfcUfgGfaGfuUfuAfuU  935 P*uCfcGfaAfuA  936 AD-15084fcGfgAfTsT faAfcUfcCfaGf gCfcTsT 2743-2761 GGCfCfUfGGAGUfUfUfAUfU  937UfCfCfGAAUfAA  938 AD-15094 fCfGGATsT ACfUfCfCfAGGC fCfTsT 2743-2761GgCcUgGaGuUuAuUcGgATsT  939 P*uCfcGfaAfuA  940 AD-15104 faAfcUfcCfaGfgCfcTsT 2743-2761 GgCcUgGaGuUuAuUcGgATsT  941 UfCfCfGAAUfAA  942AD-15114 ACfUfCfCfAGGC fCfTsT 2743-2761 GfgCfcUfgGfaGfuUfuAfuU  943UCCGAauAAacUC  944 AD-15124 fcGfgAfTsT CAGgccTsT 2743-2761GGCfCfUfGGAGUfUfUfAUfU  945 UCCGAauAAacUC  946 AD-15134 fCfGGATsTCAGgccTsT 2743-2761 GgCcUgGaGuUuAuUcGgATsT  947 UCCGAauAAacUC  948AD-15144 CAGgccTsT 2753-2771 GCAGGAUUCUUCCCAUGGATT  949 UCCAUGGGAAGAA 950 AD-15391 UCCUGCTT 2794-2812 UGCAGGGACAAACAUCGUUTT  951AACGAUGUUUGUC  952 AD-15348 CCUGCATT 2795-2813 GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGU  954 AD-15349 CCCUGCTT 2797-2815AGGGACAAACAUCGUUGGGTT  955 CCCAACGAUGUUU  956 AD-15170 GUCCCUTT2841-2859 CCCUCAUCUCCAGCUAACUTT  957 AGUUAGCUGGAGA  958 AD-15350UGAGGGTT 2845-2863 CAUCUCCAGCUAACUGUGGTT  959 CCACAGUUAGCUG  960AD-15402 GAGAUGTT 2878-2896 GCUCCCUGAUUAAUGGAGGTT  961 CCUCCAUUAAUCA 962 AD-15293 GGGAGCTT 2881-2899 CCCUGAUUAAUGGAGGCUUTT  963AAGCCUCCAUUAA  964 AD-15351 UCAGGGTT 2882-2900 CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUA  966 AD-15403 AUCAGGTT 2884-2902UGAUUAAUGGAGGCUUAGCTT  967 GCUAAGCCUCCAU  968 AD-15404 UAAUCATT2885-2903 GAUUAAUGGAGGCUUAGCUTT  969 AGCUAAGCCUCCA  970 AD-15207UUAAUCTT 2886-2904 AUUAAUGGAGGCUUAGCUUTT  971 AAGCUAAGCCUCC  972AD-15352 AUUAAUTT 2887-2905 UUAAUGGAGGCUUAGCUUUTT  973 AAAGCUAAGCCUC 974 AD-15255 CAUUAATT 2903-2921 UUUCUGGAUGGCAUCUAGCTsT  975GCUAGAUGCCAUC  976 AD-9603 CAGAAATsT 2903-2921 uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUC  978 AD-9729 cAGAAATsT 2904-2922UUCUGGAUGGCAUCUAGCCTsT  979 GGCUAGAUGCCAU  980 AD-9599 CCAGAATsT2904-2922 uucuGGAuGGcAucuAGccTsT  981 GGCuAGAUGCcAU  982 AD-9725CcAGAATsT 2905-2923 UCUGGAUGGCAUCUAGCCATsT  983 UGGCUAGAUGCCA  984AD-9621 UCCAGATsT 2905-2923 ucuGGAuGGcAucuAGccATsT  985 UGGCuAGAUGCcA 986 AD-9747 UCcAGATsT 2925-2943 AGGCUGGAGACAGGUGCGCTT  987GCGCACCUGUCUC  988 AD-15405 CAGCCUTT 2926-2944 GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCU  990 AD-15353 CCAGCCTT 2927-2945GCUGGAGACAGGUGCGCCCTT  991 GGGCGCACCUGUC  992 AD-15354 UCCAGCTT2972-2990 UUCCUGAGCCACCUUUACUTT  993 AGUAAAGGUGGCU  994 AD-15406CAGGAATT 2973-2991 UCCUGAGCCACCUUUACUCTT  995 GAGUAAAGGUGGC  996AD-15407 UCAGGATT 2974-2992 CCUGAGCCACCUUUACUCUTT  997 AGAGUAAAGGUGG 998 AD-15355 CUCAGGTT 2976-2994 UGAGCCACCUUUACUCUGCTT  999GCAGAGUAAAGGU 1000 AD-15356 GGCUCATT 2978-2996 AGCCACCUUUACUCUGCUCTT1001 GAGCAGAGUAAAG 1002 AD-15357 GUGGCUTT 2981-2999CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUA 1004 AD-15269 AAGGUGTT2987-3005 UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGC 1006 AD-9565AGAGUATsT 2987-3005 uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGc 1008AD-9691 AGAGuATsT 2998-3016 AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCC1010 AD-15358 UGGCAUTT 3003-3021 AGGCUGUGCUAGCAACACCTT 1011GGUGUUGCUAGCA 1012 AD-15359 CAGCCUTT 3006-3024 CUGUGCUAGCAACACCCAATT1013 UUGGGUGUUGCUA 1014 AD-15360 GCACAGTT 3010-3028GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUU 1016 AD-15219 GCUAGCTT3038-3056 GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAU 1018 AD-15361GGCUCCTT 3046-3064 CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCC 1020AD-15273 UAGGUGTT 3051-3069 AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUC1022 AD-15362 AGUCCUTT 3052-3070 GGACUGACUCGGCAGUGUGTT 1023CACACUGCCGAGU 1024 AD-15192 CAGUCCTT 3074-3092 UGGUGCAUGCACUGUCUCATT1025 UGAGACAGUGCAU 1026 AD-15256 GCACCATT 3080-3098AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACA 1028 AD-15363 GUGCAUTT3085-3103 CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUG 1030 AD-15364AGACAGTT 3089-3107 CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUG 1032AD-9604 GCUGAGTsT 3089-3107 cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUG1034 AD-9730 GCUGAGTsT 3093-3111 GCCAACCCGCUCCACUACCTsT 1035GGUAGUGGAGCGG 1036 AD-9527 GUUGGCTsT 3093-3111 GccAAcccGcuccAcuAccTsT1037 GGuAGUGGAGCGG 1038 AD-9653 GUUGGCTsT 3096-3114AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAG 1040 AD-15365 CGGGUUTT3099-3117 CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUG 1042 AD-15294GAGCGGTT 3107-3125 CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCC 1044AD-15173 GGGUAGTT 3108-3126 UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGC1046 AD-15366 CGGGUATT 3109-3127 ACCCGGCAGGGUACACAUUTT 1047AAUGUGUACCCUG 1048 AD-15367 CCGGGUTT 3110-3128 CCCGGCAGGGUACACAUUCTT1049 GAAUGUGUACCCU 1050 AD-15257 GCCGGGTT 3112-3130CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACC 1052 AD-15184 CUGCCGTT3114-3132 GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUA 1054 AD-15185CCCUGCTT 3115-3133 CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGU 1056AD-15258 ACCCUGTT 3116-3134 AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUG1058 AD-15186 UACCCUTT 3196-3214 GGAACUGAGCCAGAAACGCTT 1059GCGUUUCUGGCUC 1060 AD-15274 AGUUCCTT 3197-3215 GAACUGAGCCAGAAACGCATT1061 UGCGUUUCUGGCU 1062 AD-15368 CAGUUCTT 3198-3216AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGC 1064 AD-15369 UCAGUUTT3201-3219 UGAGCCAGAAACGCAGAUUTT 1064 AAUCUGCGUUUCU 1066 AD-15370GGCUCATT 3207-3225 AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGC 1068AD-15259 GUUUCUTT 3210-3228 AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUC1070 AD-15408 UGCGUUTT 3233-3251 AGCCAAGCCUCUUCUUACUTsT 1071AGUAAGAAGAGGC 1072 AD-9597 UUGGCUTsT 3233-3251 AGccAAGccucuucuuAcuTsT1073 AGuAAGAAGAGGC 1074 AD-9723 UUGGCUTsT 3233-3251AfgCfcAfaGfcCfuCfuUfcU 1075 P*aGfuAfaGfaA 1076 AD-14680 fuAfcUfTsTfgAfgGfcUfuGf gCfuTsT 3233-3251 AGCfCfAAGCfCfUfCfUfUfC 1077AGUfAAGAAGAGG 1078 AD-14690 fUfUfACfUfTsT CfUfUfGGCfUfT sT 3233-3251AgCcAaGcCuCuUcUuAcUTsT 1079 P*aGfuAfaGfaA 1080 AD-14700 fgAfgGfcUfuGfgCfuTsT 3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGG 1082AD-14710 CfUfUfGGCfUfT sT 3233-3251 AfgCfcAfaGfcCfuCfuUfcU 1083AGUAAgaAGagGC 1084 AD-14720 fuAfcUfTsT UUGgcuTsT 3233-3251AGCfCfAAGCfCfUfCfUfUfC 1085 AGUAAgaAGagGC 1086 AD-14730 fUfUfACfUfTsTUUGgcuTsT 3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGC 1088AD-14740 UUGgcuTsT 3233-3251 UfgGfuUfcCfcUfgAfgGfaC 1089 P*gCfuGfgUfcC1090 AD-15086 fcAfgCfTsT fuCfaGfgGfaAf cCfaTsT 3233-3251UfGGUfUfCfCfCfUfGAGGAC 1091 GCfUfGGUfCfCf 1092 AD-15096 fCfAGCfTsTUfCfAGGGAACfC fATsT 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1093 P*gCfuGfgUfcC1094 AD-15106 fuCfaGfgGfaAf cCfaTsT 3233-3251 UgGuUcCcUgAgGaCcAgCTsT1095 GCfUfGGUfCfCf 1096 AD-15116 UfCfAGGGAACfC fATsT 3233-3251UfgGfuUfcCfcUfgAfgGfaC 1097 GCUGGucCUcaGG 1098 AD-15126 fcAfgCfTsTGAAccaTsT 3233-3251 UfGGUfUfCfCfCfUfGAGGAC 1099 GCUGGucCUcaGG 1100AD-15136 fCfAGCfTsT GAAccaTsT 3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1101GCUGGucCUcaGG 1102 AD-15146 GAAccaTsT 3242-3260 UCUUCUUACUUCACCCGGCTT1103 GCCGGGUGAAGUA 1104 AD-15260 AGAAGATT 3243-3261CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGU 1106 AD-15371 AAGAAGTT3244-3262 UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAG 1108 AD-15372UAAGAATT 3262-3280 GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAG 1110AD-15172 GAGCCCTT 3263-3281 GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGA1112 AD-15295 GGAGCCTT 3264-3282 GCUCCUCAUUUUUACGGGUTT 1113ACCCGUAAAAAUG 1114 AD-15373 AGGAGCTT 3265-3283 CUCCUCAUUUUUACGGGUATT1115 UACCCGUAAAAAU 1116 AD-15163 GAGGAGTT 3266-3284UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAA 1118 AD-15165 UGAGGATT3267-3285 CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAA 1120 AD-15374AUGAGGTT 3268-3286 CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAA 1122AD-15296 AAUGAGTT 3270-3288 CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUA1124 AD-15261 AAAAUGTT 3271-3289 AUUUUUACGGGUAACAGUGTT 1125CACUGUUACCCGU 1126 AD-15375 AAAAAUTT 3274-3292 UUUACGGGUAACAGUGAGGTT1127 CCUCACUGUUACC 1128 AD-15262 CGUAAATT 3308-3326CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCU 1130 AD-15376 GGUCUGTT3310-3328 GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUC 1132 AD-15377CUGGUCTT 3312-3330 CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCU 1134AD-15409 UCCUGGTT 3315-3333 GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGA1136 AD-15378 GCUUCCTT 3324-3342 GUGAGUGAUGGCAGAACGATT 1137UCGUUCUGCCAUC 1138 AD-15410 ACUCACTT 3326-3344 GAGUGAUGGCAGAACGAUGTT1139 CAUCGUUCUGCCA 1140 AD-15379 UCACUCTT 3330-3348GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCU 1142 AD-15187 GCCAUCTT3336-3354 AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAU 1144 AD-15263CGUUCUTT 3339-3357 ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGG 1146AD-15264 CAUCGUTT 3348-3366 GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAU1148 AD-15297 GCCUGCTT 3356-3374 GGAACUUUUUCCGUUAUCATT 1149UGAUAACGGAAAA 1150 AD-15208 AGUUCCTT 3357-3375 GAACUUUUUCCGUUAUCACTT1151 GUGAUAACGGAAA 1152 AD-15209 AAGUUCTT 3358-3376AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAA 1154 AD-15193 AAAGUUTT3370-3388 UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGG 1156 AD-15380GUGAUATT 3378-3396 AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUC 1158AD-15298 AGGCCUTT 3383-3401 UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGU1160 AD-15299 GAAUCATT 3385-3403 AUUCACUGGCCUGGCGGAGTT 1161CUCCGCCAGGCCA 1162 AD-15265 GUGAAUTT 3406-3424 GCUUCUAAGGCAUGGUCGGTT1163 CCGACCAUGCCUU 1164 AD-15381 AGAAGCTT 3407-3425CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCU 1166 AD-15210 UAGAAGTT3429-3447 GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUG 1168 AD-15270GCCCUCTT 3440-3458 ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGG 1170AD-9591 GACAGUTsT 3440-3458 AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGG1172 AD-9717 GAcAGUTsT 3441-3459 CUGUCCCUCCUUGAGCACCTsT 1173GGUGCUCAAGGAG 1174 AD-9622 GGACAGTsT 3441-3459 cuGucccuccuuGAGcAccTsT1175 GGUGCUcAAGGAG 1176 AD-9748 GGAcAGTsT 3480-3498ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAU 1178 AD-9587 AAAUGUTsT3480-3498 AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAu 1180 AD-9713AAAUGUTsT 3480-3498 AfcAfuUfuAfuCfuUfuUfgG 1181 P*aGfaCfcCfaA 1182AD-14679 fgUfcUfTsT faAfgAfuAfaAf uGfuTsT 3480-3498ACfAUfUfUfAUfCfUfUfUfU 1183 AGACfCfCfAAAA 1184 AD-14689 fGGGUfCfUfTsTGAUfAAAUfGUfT sT 3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1185 P*aGfaCfcCfaA1186 AD-14699 faAfgAfuAfaAf uGfuTsT 3480-3498 AcAuUuAuCuUuUgGgUcUTsT1187 AGACfCfCfAAAA 1188 AD-14709 GAUfAAAUfGUfT sT 3480-3498AfcAfuUfuAfuCfuUfuUfgG 1189 AGACCcaAAagAU 1190 AD-14719 fgUfcUfTsTAAAuguTsT 3480-3498 ACfAUfUfUfAUfCfUfUfUfU 1191 AGACCcaAAagAU 1192AD-14729 fGGGUfCfUfTsT AAAuguTsT 3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1193AGACCcaAAagAU 1194 AD-14739 AAAuguTsT 3480-3498 GfcCfaUfcUfgCfuGfcCfgG1195 P*gGfcUfcCfgG 1196 AD-15085 faGfcCfTsT fcAfgCfaGfaUf gGfcTsT3480-3498 GCfCfAUfCfUfGCfUfGCfCf 1197 GGCfUfCfCfGGC 1198 AD-15095GGAGCfCfTsT fAGCfAGAUfGGC fTsT 3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1199P*gGfcUfcCfgG 1200 AD-15105 fcAfgCfaGfaUf gGfcTsT 3480-3498GcCaUcUgCuGcCgGaGcCTsT 1201 GGCfUfCfCfGGC 1202 AD-15115 fAGCfAGAUfGGCfTsT 3480-3498 GfcCfaUfcUfgCfuGfcCfgG 1203 GGCUCauGCagCA 1204 AD-15125faGfcCfTsT GAUggcTsT 3480-3498 GCfCfAUfCfUfGCfUfGCfCf 1205 GGCUCauGCagCA1206 AD-15135 GGAGCfCfTsT GAUggcTsT 3480-3498 GcCaUcUgCuGcCgGaGcCTsT1207 GGCUCauGCagCA 1208 AD-15145 GAUggcTsT 3481-3499CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGA 1210 AD-9578 UAAAUGTsT3481-3499 cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGA 1212 AD-9704uAAAUGTsT 3485-3503 UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAA 1214AD-9558 AAGAUATsT 3485-3503 uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAA1216 AD-9684 AAGAuATsT 3504-3522 CUCUGUUGCCUUUUUACAGTsT 1217CUGUAAAAAGGCA 1218 AD-9634 ACAGAGTsT 3504-3522 cucuGuuGccuuuuuAcAGTsT1219 CUGuAAAAAGGcA 1220 AD-9760 AcAGAGTsT 3512-3530CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUA 1222 AD-15411 AAAAGGTT3521-3539 AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAG 1224 AD-15266UUGGCUTT 3526-3544 ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAG 1226AD-15382 AAAAGUTT 3530-3548 UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGU1228 AD-9554 CUAGAATsT 3530-3548 uucuAGAccuGuuuuGcuuTsT 1229AAGcAAAAcAGGU 1230 AD-9680 CuAGAATsT 3530-3548 UfuCfuAfgAfcCfuGfuUfuU1231 P*aAfgCfaAfaA 1232 AD-14676 fgCfuUfTsT fcAfgGfuCfuAf gAfaTsT3530-3548 UfUfCfUfAGACfCfUfGUfUf 1233 AAGCfAAAACfAG 1234 AD-14686UfUfGCfUfUfTsT GUfCfUfAGAATs T 3530-3548 UuCuAgAcCuGuUuUgCuUTsT 1235P*aAfgCfaAfaA 1236 AD-14696 fcAfgGfuCfuAf gAfaTsT 3530-3548UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAG 1238 AD-14706 GUfCfUfAGAATs T3530-3548 UfuCfuAfgAfcCfuGfuUfuU 1239 AAGcAaaACagGU 1240 AD-14716ffCfuUfTsT CUAgaaTsT 3530-3548 UfUfCfUfAGACfCfUfGUfUf 1241 AAGcAaaACagGU1242 AD-14726 UfUfGCfUfUfTsT CUAgaaTsT 3530-3548 UuCuAgAcCuGuUuUgCuUTsT1243 AAGcAaaACagGU 1244 AD-14736 CUAgaaTsT 3530-3548CfaUfaGfgCfcUfgGfaGfuU 1245 P*aAfuAfaAfcU 1246 AD-15082 fuAfuUfTsTfcCfaGfgCfcUf aUfgTsT 3530-3548 CfAUfAGGCfCfUfGGAGUfUf 1247AAUfAAACfUfCf 1248 AD-15092 UfAUfUfTsT CfAGGCfCfUfAU fGTsT 3530-3548CaUaGgCcUgGaGuUuAuUTsT 1249 P*aAfuAfaAfcU 1250 AD-15102 fcCfaGfgCfcUfaUfgTsT 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCf 1252AD-15112 CfAGGCfCfUfAU fGTsT 3530-3548 CfaUfaGfgCfcUfgGfaGfuU 1253AAUAAacUCcaGG 1254 AD-15122 fuAfuUfTsT CCUaugTsT 3530-3548CfAUfAGGCfCfUfGGAGUfUf 1255 AAUAAacUCcaGG 1256 AD-15132 UfAUfUfTsTCCUaugTsT 3530-3548 CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGG 1258AD-15142 CCUaugTsT 3531-3549 UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGG1260 AD-9553 UCUAGATsT 3531-3549 ucuAGAccuGuuuuGcuuuTsT 1261AAAGcAAAAcAGG 1262 AD-9679 UCuAGATsT 3531-3549 UfcUfaGfaCfcUfgUfuUfuG1263 P*aAfaGfcAfaA 1264 AD-14675 fcUfuUfTsT faCfaGfgUfcUf aGfaTsT3531-3549 UfCfUfAGACfCfUfGUfUfUf 1265 AAAGCfAAAACfA 1266 AD-14685UfGCfUfUfUfTsT GGUfCfUfAGATs T 3531-3549 UcUaGaCcUgUuUuGcUuUTsT 1267P*aAfaGfcAfaA 1268 AD-14695 faCfaGfgUfcUf aGfaTsT 3531-3549UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfA 1270 AD-14705 GGUfCfUfAGATs T3531-3549 UfcUfaGfaCfcUfgUfuUfuG 1271 AAAGCaaAAcaGG 1272 AD-14715fcUfuUfTsT UCUagaTsT 3531-3549 UfCfUfAGACfCfUfGUfUfUf 1273 AAAGCaaAAcaGG1274 AD-14725 UfGCfUfUfUfTsT UCUagaTsT 3531-3549 UcUaGaCcUgUuUuGcUuUTsT1275 AAAGCaaAAcaGG 1276 AD-14735 UCUagaTsT 3531-3549UfcAfuAfgGfcCfuGfgAfgU 1277 P*aUfaAfaCfuC 1278 AD-15081 fuUfaUfTsTfcAfgGfcCfuAf uGfaTsT 3531-3549 UfCfAUfAGGCfCfUfGGAGUf 1279AUfAAACfUfCfC 1280 AD-15091 UfUfAUfTsT fAGGCfCfUfAUf GATsT 3531-3549UcAuAgGcCuGgAgUuUaUTsT 1281 P*aUfaAfaCfuC 1282 AD-15101 fcAfgGfcCfuAfuGfaTsT 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAACfUfCfC 1284AD-15111 fAGGCfCfUfAUf GATsT 3531-3549 UfcAfuAfgGfcCfuGfgAfgU 1285AUAAAcuCCagGC 1286 AD-15121 fuUfaUfTsT CUAugaTsT 3531-3549UfCfAUfAGGCfCfUfGGAGUf 1287 AUAAAcuCCagGC 1288 AD-15131 UfUfAUfTsTCUAugaTsT 3531-3549 UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGC 1290AD-15141 CUAugaTsT 3557-3575 UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUA1292 AD-9626 UCUUCATsT 3557-3575 uGAAGAuAuuuAuucuGGGTsT 1293CCcAGAAuAAAuA 1294 AD-9752 UCUUcATsT 3570-3588 UCUGGGUUUUGUAGCAUUUTsT1295 AAAUGCUACAAAA 1296 AD-9629 CCCAGATsT 3570-3588ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAA 1298 AD-9755 CCcAGATsT3613-3631 AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGU 1300 AD-15412UUUUAUTT 3617-3635 AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGU 1302AD-15211 UUGUUUTT 3618-3636 AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUG1304 AD-15300 UUUGUUTT *Target: target in human PCSK9 gene, access.# NM_174936 U, C, A, G: corresponding ribonucleotide; T: deoxythymidine;u, c, a g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf:corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides arewritten in sequence, they are connected by 3′-5′ phosphodiester groups;nucleotides with interjected “s” are connected by 3′-O-5′-Ophosphorodiester groups; unless denoted by prefix “P*“, oligonucleotidesare devoid of a 5′-phosphate group on the 5′-most nucleotide; alloligonucleotides bear 3′-OH on the 3′-most nucleotide.

TABLE 5 Sequences of modified dsRNA targeted to PCSK9Sense strand sequence SEQ ID Antisense-strand SEQ ID Duplex # (5′-3′)¹NO: sequence (5′-3′)¹ NO:  AD-10792 GccuGGAGuuuAuucGGAATsT 1305UUCCGAAuAAACUCcAGGCTsT 1306 AD-10793 GccuGGAGuuuAuucGGAATsT 1307uUcCGAAuAAACUccAGGCTsT 1308 AD-10796 GccuGGAGuuuAuucGGAATsT 1309UUCCGAAUAAACUCCAGGCTsT 1310 AD-12038 GccuGGAGuuuAuucGGAATsT 1311UUCCGAAUAAACUCCAGGCTsT 1312 AD-12039 GccuGGAGuuuAuucGGAATsT 1313UUCCGAAUAAACUCCAGGCTsT 1314 AD-12040 GccuGGAGuuuAuucGGAATsT 1315UUcCGAAUAAACUCCAGGCTsT 1316 AD-12041 GccuGGAGuuuAuucGGAATsT 1317UUCCGAAUAAACUCCAGGCTsT 1318 AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319UUCCGAAUAAACUCCAGGCTsT 1320 AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321UUCCGAAUAAACUCCAGGCTsT 1322 AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323UUCCGAAUAAACUCCAGGCTsT 1324 AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325UUCCGAAUAAACUCCAGGCTsT 1326 AD-12046 GccuGGAGuuuAuucGGAA 1327UUCCGAAUAAACUCCAGGCscsu 1328 AD-12047 GccuGGAGuuuAuucGGAAA 1329UUUCCGAAUAAACUCCAGGCscsu 1330 AD-12048 GccuGGAGuuuAuuGGAAAA 1331UUUUCCGAAUAAACUCCAGGCscsu 1332 AD-12049 GccuGGAGuuuAuucGGAAAAG 1333CUUUUCCGAAUAAACUCCAGGCscsu 1334 AD-12050 GccuGGAGuuuAuucGGAATTab 1335UUCCGAAUAAACUCCAGGCTTab 1336 AD-12051 GccuGGAGuuuAuucGGAAATTab 1337UUUCCGAAUAAACUCCAGGCTTab 1338 AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339UUUUCCGAAUAAACUCCAGGCTTab 1340 AD-12053 GccuGGAGuuuAuucGGAAAAGTTab 1341CUUUUCCGAAUAAACUCCAGGCTTab 1342 AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343UUCCGAAUAAACUCCAGGCscsu 1344 AD-12055 GccuGGAGuuuAuucGGAATsT 1345UUCCGAAUAAACUCCAGGCscsu 1346 AD-12056 GcCuGgAgUuUaUuCgGaA 1347UUCCGAAUAAACUCCAGGCTTab 1348 AD-12057 GcCuGgAgUuUaUuCgGaA 1349UUCCGAAUAAACUCCAGGCTsT 1350 AD-12058 GcCuGgAgUuUaUuCgGaA 1351UUCCGAAUAAACUCCAGGCTsT 1352 AD-12059 GcCuGgAgUuUaUuCgGaA 1353uUcCGAAuAAACUccAGGCTsT 1354 AD-12060 GcCuGgAgUuUaUuCgGaA 1355UUCCGaaUAaaCUCCAggc 1356 AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357UUCCGaaUAaaCUCCAggcTsT 1358 AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359UUCCGaaUAaaCUCCAggcTTab 1360 AD-12063 GcCuGgAgUuUaUuCgGaA 1361UUCCGaaUAaaCUCCAggcscsu 1362 AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363UUCCGAAuAAACUCcAGGCTsT 1364 AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365UUCCGAAuAAACUCcAGGCTTab 1366 AD-12066 GcCuGgAgUuUaUuCgGaA 1367UUCCGAAuAAACUCcAGGCscsu 1368 AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369UUCCGAAUAAACUCCAGGCTsT 1370 AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371UUCCGAAUAAACUCCAGGCTTab 1372 AD-12069 GcCuGgAgUuUaUuCgGaA 1373UUCCGAAUAAACUCCAGGCScsu 1374 AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1375P*uUfcCfgAfaUfaAfaCf 1376 uCfcAfgGfc AD-12339 GcCuGgAgUuUaUuCgGaA 1377P*uUfcCfgAfaUfaAfaCf 1378 uCfcAfgGfc AD-12340 GccuGGAGuuuAuucGGAA 1379P*uUfcCfgAfaUfaAfaCf 1380 uCfcAfgGfc AD-12341GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1381 P*uUfcCfgAfaUfaAfaCf 1382uCfcAfgGfcTsT AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1383UUCCGAAuAAACUCcAGGCTsT 1384 AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT1385 uUcCGAAuAAACUccAGGCTsT 1386 AD-12344GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1389 UUCCGAAUAAACUCCAGGCScsu1390 AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1391UUCCGaaUAaaCUCCAggcscsu 1392 AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393P*uUfcCfgAfaUfaAfaCf 1394 uCfcAfgGfcTsT AD-12348 GccuGGAGuuuAuucGGAATsT1395 P*uUfcCfgAfaUfaAfaCf 1396 uCfcAfgGfcTsT AD-12349GcCuGgnAgUuUaUuCgGaATsT 1397 P*uUfcCfgAfaUfaAfaCf 1398 uCfcAfgGfcTsTAD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab 1399 P*uUfcCfgAfaUfaAfaCf1400 uCfcAfgGfcTTab AD-12351 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1401P*uUfcCfgAfaUfaAfaCf 1402 uCfcAfgGfcsCfsu AD-12352GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1403 UUCCGaaUAaaCUCCAggcs 1404 csuAD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1405 UUCCGAAUAAACUCCAGGCs 1406csu AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1407 UUCCGAAUAAACUCCAGGCT1408 sT AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1409 uUcCGAAuAAACUccAGGCT1410 sT AD-12357 GmocCmouGmogAm02gUmouUmoaUmo 1411 UUCCGaaUAaaCUCCAggc1412 uCmogGmoaA AD-12358 GmocCmouGmogAm02gUmouUmoaUmo 1413P*uUfcCfgAfaUfaAfaCf 1414 uCmogGmoaA uCfcAfgGfc AD-12359GmocCmouGmogAm02gUmouUmoaUmo 1415 P*uUfcCfgAfaUfaAfaCf 1416 uCmogGmoaAuCfcAfgGfcsCfsu AD-12360 GmocCmouGmogAm02gUmouUmoaUmo 1417UUCCGAAUAAACUCCAGGCS 1418 uCmogGmoaA csu AD-12361GmocCmouGmogAm02gUmouUmoaUmo 1419 UUCCGAAuAAACUCcAGGCT 1420 uCmogGmoaAsT AD-12362 GmocCmouGmogAm02gUmouUmoaUmo 1421 uUcCGAAuAAACUccAGGCT 1422uCmogGmoaA sT AD-12363 GmocCmouGmogAm02gUmouUmoaUmo 1423UUCCGaaUAaaCUCCAggcs 1424 uCmogGmoaA csu AD-12364GmocCmouGmogAmogUmouUmoaUmou 1425 UUCCGaaUAaaCUCCAggcT 1426 CmogGmoaATsTsT AD-12365 GmocCmouGmogAmogUmouUmoaUmou 1427 UUCCGAAuAAACUCcAGGCT 1428CmogGmoaATsT sT AD-12366 GmocCmouGmogAmogUmouUmoaUmou 1429UUCCGAAUAAACUCCAGGCT 1430 CmogGmoaATsT sT AD-12367GmocmocmouGGAGmoumoumouAmoum 1431 UUCCGaaUAaaCUCCAggcT 1432 oumocGGAATsTsT AD-12368 GmocmocmouGGAGmoumoumouAmoum 1433 UUCCGAAuAAACUCcAGGCT 1434oumocGGAATsT sT AD-12369 GmocmocmouGGAGmoumoumouAmoum 1435UUCCGAAUAAACUCCAGGCT 1436 oumocGGAATsT sT AD-12370GmocmocmouGGAGmoumoumouAmoum 1437 P*UfUfCfCfGAAUfAAACf 1438 oumocGGAATsTUfCfCfAGGCfTsT AD-12371 GmocmocmouGGAGmoumoumouAmoum 1439P*UfUfCfCfGAAUfAAACf 1440 oumocGGAATsT UfCfCfAGGCfsCfsUf AD-12372GmocmocmouGGAGmoumoumouAmoum 1441 P*uUfcCfgAfaUfaAfaCf 1442 oumocGGAATsTuCfcAfgGfcsCfsu AD-12373 GmocmocmouGGAGmoumoumouAmoum 1443UUCCGAAUAAACUCCAGGCT 1444 oumocGGAATsT sT AD-12374GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1445 UfUfCfCfGAAUfAAACfUf 1446 TsTCfCfAGGCfTsT AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1447UUCCGAAUAAACUCCAGGCT 1448 TsT sT AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAA1449 uUcCGAAuAAACUccAGGCT 1450 TsT sT AD-12378GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1451 UUCCGaaUAaaCUCCAggcs 1452 TsT csuAD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1453 UUCCGAAUAAACUCCAGGCS 1454 TsTcsu AD-12380 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1455 P*uUfcCfgAfaUfaAfaCf 1456TsT uCfcAfgGfcsCfsu AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1457P*uUfcCfgAfaUfaAfaCf 1458 TsT uCfcAfgGfcTsT AD-12382GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1459 P*UfUfCfCfGAAUfAAACf 1460 TsTUfCfCfAGGCfTsT AD-12383 GCCUGGAGUUUAUUCGGAATST 1461 P*UfUfCfCfGAAUfAAACf1462 UfCfCfAGGCfTsT AD-12384 GccuGGAGuuuAuucGGAATsT 1463P*UfUfCfCfGAAUfAAACf 1464 UfCfCfAGGCfTsT AD-12385GcCuGgnAgUuUaUuCgGaATsT 1465 P*UfUfCfCfGAAUfAAACf 1466 UfCfCfAGGCfTsTAD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1467 P*UfUfCfCfGAAUfAAACf 1468UfCfCfAGGCfTsT AD-12387 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1469UfUfCfCfGAAUfAAACfUf 1470 CfCfAGGCfsCfsUf AD-12388GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1471 P*uUfcCfgAfaUfaAfaCf 1472 uCfcAfgGfcAD-12389 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1473 P*uUfcCfgAfaUfaAfaCf 1474uCfcAfgGfcsCfsu AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475UUCCGAAUAAACUCCAGGCS 1476 csu AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1477 UUCCGaaUAaaCUCCAggc 1478 AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1479 UUCCGAAUAAACUCCAGGCT 1480 sT AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1481 UUCCGAAuAAACUCcAGGCT 1482 sT AD-12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA1483 uUcCGAAuAAACUccAGGCT 1484 sT AD-12395 GmocCmouGmogAmogUmouUmoaUmou1485 P*UfUfCfCfGAAUfAAACf 1486 CmogGmoaATsT UfCfCfAGGCfsCfsUf AD-12396GmocCmouGmogAm02gUmouUmoaUmo 1487 P*UfUfCfCfGAAUfAAACf 1488 uCmogGmoaAUfCfCfAGGCfsCfsUf AD-12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489P*UfUfCfCfGAAUfAAACf 1490 UfCfCfAGGCfsCfsUf AD-12398GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1491 P*UfUfCfCfGAAUfAAACf 1492UfCfCfAGGCfsCfsUf AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493P*UfUfCfCfGAAUfAAACf 1494 UfCfCfAGGCfsCfsUf AD-12400GCCUGGAGUUUAUUCGGAATST 1495 P*UfUfCfCfGAAUfAAACf 1496 UfCfCfAGGCfsCfsUfAD-12401 GccuGGAGuuuAuucGGAATsT 1497 P*UfUfCfCfGAAUfAAACf 1498UfCfCfAGGCfsCfsUf AD-12402 GccuGGAGuuuAuucGGAA 1499 P*UfUfCfCfGAAUfAAACf1500 UfCfCfAGGCfsCfsUf AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501P*UfUfCfCfGAAUfAAACf 1502 UfCfCfAGGCfsCfsUf AD-9314GCCUGGAGUUUAUUCGGAATST 1503 UUCCGAAUAAACUCCAGGCT 1504 sT AD-10794ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAd 1526 TsdT AD-10795ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAd 1528 TsdT AD-10797ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAd 1530 TsdT  U, C, A,G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written insequence, they are connected by 3′-5′ phosphodiester groups; nucleotideswith inteijected “s” are connected by 3′-O-5′-O phosphorothiodiestergroups; unless denoted by prefix “P*”, oligonucleotides are devoid of a5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear3′-OH on the 3′-most nucleotide.

TABLE 6  dsRNA targeted to PCSK9: mismatches and modifications Duplex #Strand SEQ ID NO: Sequence (5′ to 3′)  AD-9680 S 1531uucuAGAccuGuuuuGcuudTsdT AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT AD-3267 S 1535uucuAGAcCuGuuuuGcuuTsT AS 1536 AAGcAAAAcAGGUCuAGAATsT AD-3268 S 1537uucuAGAccUGuuuuGcuuTsT AS 1538 AAGcAAAAcAGGUCuAGAATsT AD-3269 S 1539uucuAGAcCUGuuuuGcuuTsT AS 1540 AAGcAAAAcAGGUCuAGAATsT AD-3270 S 1541uucuAGAcY1uGuuuuGcuuTsT AS 1542 AAGcAAAAcAGGUCuAGAATsT AD-3271 S 1543uucuAGAcY1UGuuuuGcuuTsT AS 1544 AAGcAAAAcAGGUCuAGAATsT AD-3272 S 1545uucuAGAccYIGuuuuGcuuTsT AS 1546 AAGcAAAAcAGGUCuAGAATsT AD-3273 S 1547uucuAGAcCY1GuuuuGcuuTsT AS 1548 AAGcAAAAcAGGUCuAGAATsT AD-3274 S 1549uucuAGAccuY1uuuuGcuuTsT AS 1550 AAGcAAAAcAGGUCuAGAATsT AD-3275 S 1551uucuAGAcCUY1uuuuGcuuTsT AS 1552 AAGcAAAAcAGGUCuAGAATsT AD-14676 S 1553UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT AS 1554P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3276 S 1555UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT AS 1556P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3277 S 1557UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT AS 1558P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3278 S 1559UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT AS 1560P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3279 S 1561UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT AS 1562P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3280 S 1563UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT AS 1564P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3281 S 1565UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT AS 1566P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3282 S 1567UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT AS 1568P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3283 S 1569UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT AS 1570P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3284 S 1571UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT AS 1572P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-10792 S 459 GccuGGAGuuuAuucGGAATsTAS 460 UUCCGAAuAAACUCcAGGCTsT AD-3254 S 1573 GccuGGAGuYIuAuucGGAATsT AS1574 UUCCGAAuAAACUCcAGGCTsT AD-3255 S 1575 GccuGGAGUY1uAuucGGAATsT AS1576 UUCCGAAuAAACUCcAGGCTsT  Strand: S/Sense; AS/Antisense; U, C, A, G:corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af. Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluylribo(or deoxyribo)nucleotide; where nucleotides are written in sequence,they are connected by 3′-5′ phosphodiester groups; nucleotides withinterjected “s” are connected by 3′-O-5 -O phosphorothiodiester groups;unless denoted by prefix “P*”, oligonucleotides are devoid of a5′-phospltate group on the 5′-most nucleotide; all oligonucleotides bear3′-OH on the 3′-most nucleotide

TABLE 7  Sequences of unmodified siRNA flanking AD-9680 SEQ Duplex #Strand Sequence (5′ to 3′) *Target ID NO: AD-22169-b1 senseCAGCCAACUUUUCUAGACCdTsdT 3520 1577 antis GGUCUAGAAAAGUUGGCUGdTsdT 35201578 AD-22170-b1 sense AGCCAACUUUUCUAGACCUdTsdT 3521 1579 antisAGGUCUAGAAAAGUUGGCUdTsdT 3521 1580 AD-22171-b1 senseGCCAACUUUUCUAGACCUGdTsdT 3522 1581 antis CAGGUCUAGAAAAGUUGGCdTsdT 35221582 AD-22172-b1 sense CCAACUUUUCUAGACCUGUdTsdT 3523 1583 antisACAGGUCUAGAAAAGUUGGdTsdT 3523 1584 AD-22173-b1 senseCAACUUUUCUAGACCUGUUdTsdT 3524 1585 antis AACAGGUCUAGAAAAGUUGdTsdT 35241586 AD-22174-b1 sense AACUUUUCUAGACCUGUUUdTsdT 3525 1587 antisAAACAGGUCUAGAAAAGUUdTsdT 3525 1588 AD-22175-b1 senseACUUUUCUAGACCUGUUUUdTsdT 3526 1589 antis AAAACAGGUCUAGAAAAGUdTsdT 35261590 AD-22176-b1 sense CUUUUCUAGACCUGUUUUGdTsdT 3527 1591 antisCAAAACAGGUCUAGAAAAGdTsdT 3527 1592 AD-22177-b1 senseUUUUCUAGACCUGUUUUGCdTsdT 3528 1593 antis GCAAAACAGGUCUAGAAAAdTsdT 35281594 AD-22178-b1 sense UUUCUAGACCUGUUUUGCUdTsdT 3529 1595 antisAGCAAAACAGGUCUAGAAAdTsdT 3529 1596 AD-22179-b1 senseUCUAGACCUGUUUUGCUUUdTsdT 3531 1597 antis AAAGCAAAACAGGUCUAGAdTsdT 35311598 AD-22180-b1 sense CUAGACCUGUUUUGCUUUUdTsdT 3532 1599 antisAAAAGCAAAACAGGUCUAGdTsdT 3532 1600 AD-22181-b1 senseUAGACCUGUUUUGCUUUUGdTsdT 3533 1601 antis CAAAAGCAAAACAGGUCUAdTsdT 35331602 AD-22182-b1 sense AGACCUGUUUUGCUUUUGUdTsdT 3534 1603 antisACAAAAGCAAAACAGGUCUdTsdT 3534 1604 AD-22183-b1 senseGACCUGUUUUGCUUUUGUAdTsdT 3535 1605 antis UACAAAAGCAAAACAGGUCdTsdT 35351606 AD-22184-b1 sense ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607 antisUUACAAAAGCAAAACAGGUdTsdT 3536 1608 AD-22185-b1 senseCCUGUUUUGCUUUUGUAACdTsdT 3537 1609 antis GUUACAAAAGCAAAACAGGdTsdT 35371610 AD-22186-b1 sense CUGUUUUGCUUUUGUAACUdTsdT 3538 1611 antisAGUUACAAAAGCAAAACAGdTsdT 3538 1612 AD-22187-b1 senseUGUUUUGCUUUUGUAACUUdTsdT 3539 1613 antis AAGUUACAAAAGCAAAACAdTsdT 35391614 AD-22188-b1 sense GUUUUGCUUUUGUAACUUGdTsdT 3540 1615 antisCAAGUUACAAAAGCAAAACdTsdT 3540 1616 AD-22189-b1 senseUUUUGCUUUUGUAACUUGAdTsdT 3541 1617 antis UCAAGUUACAAAAGCAAAAdTsdT 35411618 AD-22190-b1 sense UUUGCUUUUGUAACUUGAAdTsdT 3542 1619 antisUUCAAGUUACAAAAGCAAAdTsdT 3542 1620 AD-22191-b1 senseUUGCUUUUGUAACUUGAAGdTsdT 3543 1621 antis CUUCAAGUUACAAAAGCAAdTsdT 35431622 AD-22192-b1 sense UGCUUUUGUAACUUGAAGAdTsdT 3544 1623 antisUCUUCAAGUUACAAAAGCAdTsdT 3544 1624 AD-22193-b1 senseGCUUUUGUAACUUGAAGAUdTsdT 3545 1625 antis AUCUUCAAGUUACAAAAGCdTsdT 35451626 AD-22194-b1 sense CUUUUGUAACUUGAAGAUAdTsdT 3546 1627 antisUAUCUUCAAGUUACAAAAGdTsdT 3546 1628 AD-22195-b1 senseUUUUGUAACUUGAAGAUAUdTsdT 3547 1629 antis AUAUCUUCAAGUUACAAAAdTsdT 35471630 AD-22196-b1 sense UUUGUAACUUGAAGAUAUUdTsdT 3548 1631 antisAAUAUCUUCAAGUUACAAAdTsdT 3548 1632 AD-22197-b1 senseUUGUAACUUGAAGAUAUUUdTsdT 3549 1633 antis AAAUAUCUUCAAGUUACAAdTsdT 35491634 AD-22198-b1 sense UGUAACUUGAAGAUAUUUAdTsdT 3550 1635 antisUAAAUAUCUUCAAGUUACAdTsdT 3550 1636 AD-22199-b1 senseGUAACUUGAAGAUAUUUAUdTsdT 3551 1637 antis AUAAAUAUCUUCAAGUUACdTsdT 35511638 AD-22200-b1 sense UAACUUGAAGAUAUUUAUUdTsdT 3552 1639 antisAAUAAAUAUCUUCAAGUUAdTsdT 3552 1640 AD-22201-b1 senseAACUUGAAGAUAUUUAUUCdTsdT 3553 1641 antis GAAUAAAUAUCUUCAAGUUdTsdT 35531642 AD-22202-b1 sense ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643 antisAGAAUAAAUAUCUUCAAGUdTsdT 3554 1644 AD-22203-b1 senseCUUGAAGAUAUUUAUUCUGdTsdT 3555 1645 antis CAGAAUAAAUAUCUUCAAGdTsdT 35551646 AD-22204-b1 sense UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647 antisCCAGAAUAAAUAUCUUCAAdTsdT 3556 1648 AD-22205-b1 senseUGAAGAUAUUUAUUCUGGGdTsdT 3557 1649 antis CCCAGAAUAAAUAUCUUCAdTsdT 35571650 AD-22206-b1 sense GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651 antisACCCAGAAUAAAUAUCUUCdTsdT 3558 1652  *Target: target in human PCSK9 gene,access, # NM_174936 U, C, A, G: corresponding ribonucleotide; dT:deoxythymidine; where nucleotides are written in sequence, they areconnected by 3′-5′ phosphodiester groups: nucleotides with interjected“s” are connected by 3′-O-5′-O phosphorothiodiester groups.

TABLE 8  Sequences of modified siRNA flanking AD-9680 Duplex # StrandSequence (5′ to 3′) *Target SEQ ID NO:  AD-22098-b1 sensecAGccAAcuuuucuAGAccdTsdT 3520 1653 antis GGUCuAGAAAAGUUGGCUGdTsdT 35201654 AD-22099-b1 sense AGccAAcuuuucuAGAccudTsdT 3521 1655 antisAGGUCuAGAAAAGUUGGCUdTsdT 3521 1656 AD-22100-b1 senseGccAAcuuuucuAGAccuGdTsdT 3522 1657 antis cAGGUCuAGAAAAGUUGGCdTsdT 35221658 AD-22101-b1 sense ccAAcuuuucuAGAccuGudTsdT 3523 1659 antisAcAGGUCuAGAAAAGUUGGdTsdT 3523 1660 AD-22102-b1 sensecAAcuuuucuAGAccuGuudTsdT 3524 1661 antis AAcAGGUCuAGAAAAGUUGdTsdT 35241662 AD-22103-b1 sense AAcuuuucuAGAccuGuuudTsdT 3525 1663 antisAAAcAGGUCuAGAAAAGUUdTsdT 3525 1664 AD-22104-b1 senseAcuuuucuAGAccuGuuuudTsdT 3526 1665 antis AAAAcAGGUCuAGAAAAGUdTsdT 35261666 AD-22105-b1 sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667 antiscAAAAcAGGUCuAGAAAAGdTsdT 3527 1668 AD-22106-b1 senseuuuucuAGAccuGuuuuGcdTsdT 3528 1669 antis GcAAAAcAGGUCuAGAAAAdTsdT 35281670 AD-22107-b1 sense uuucuAGAccuGuuuuGcudTsdT 3529 1671 antisAGcAAAAcAGGUCuAGAAAdTsdT 3529 1672 AD-22108-b1 senseucuAGAccuGuuuuGcuuudTsdT 3531 1673 antis AAAGcAAAAcAGGUCuAGAdTsdT 35311674 AD-22109-b1 sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675 antisAAAAGcAAAAcAGGUCuAGdTsdT 3532 1676 AD-22110-b1 senseuAGAccuGuuuuGcuuuuGdTsdT 3533 1677 antis cAAAAGcAAAAcAGGUCuAdTsdT 35331678 AD-22111-b1 sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679 antisAcAAAAGcAAAAcAGGUCUdTsdT 3534 1680 AD-22112-b1 senseGAccuGuuuuGcuuuuGuAdTsdT 3535 1681 antis uAcAAAAGcAAAAcAGGUCdTsdT 35351682 AD-22113-b1 sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683 antisUuAcAAAAGcAAAAcAGGUdTsdT 3536 1684 AD-22114-b1 senseccuGuuuuGcuuuuGuAAcdTsdT 3537 1685 antis GUuAcAAAAGcAAAAcAGGdTsdT 35371686 AD-22115-b1 sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687 antisAGUuAcAAAAGcAAAAcAGdTsdT 3538 1688 sense uGuuuuGcuuuuGuAAcuudTsdT 35391689 antis AAGUuAcAAAAGcAAAAcAdTsdT 3539 1690 AD-22116-b1 senseGuuuuGcuuuuGuAAcuuGdTsdT 3540 1691 antis cAAGUuAcAAAAGcAAAACdTsdT 35401692 AD-22117-b1 sense uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693 antisUcAAGUuAcAAAAGcAAAAdTsdT 3541 1694 AD-22118-b1 senseuuuGcuuuuGuAAcuuGAAdTsdT 3542 1695 antis UUcAAGUuAcAAAAGcAAAdTsdT 35421696 AD-22119-b1 sense uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697 antisCUUcAAGUuAcAAAAGcAAdTsdT 3543 1698 AD-22120-b1 senseuGcuuuuGuAAcuuGAAGAdTsdT 3544 1699 antis UCUUcAAGUuAcAAAAGcAdTsdT 35441700 AD-22121-b1 sense GcuuuuGuAAcuuGAAGAudTsdT 3545 1701 antisAUCUUcAAGUuAcAAAAGCdTsdT 3545 1702 AD-22122-b1 sensecuuuuGuAAcuuGAAGAuAdTsdT 3546 1703 antis uAUCUUcAAGUuAcAAAAGdTsdT 35461704 AD-22123-b1 sense uuuuGuAAcuuGAAGAuAudTsdT 3547 1705 antisAuAUCUUcAAGUuAcAAAAdTsdT 3547 1706 AD-22124-b1 senseuuuGuAAcuuGAAGAuAuudTsdT 3548 1707 antis AAuAUCUUcAAGUuAcAAAdTsdT 35481708 AD-22125-b1 sense uuGuAAcuuGAAGAuAuuudTsdT 3549 1709 antisAAAuAUCUUcAAGUuAcAAdTsdT 3549 1710 AD-22126-b1 senseuGuAAcuuGAAGAuAuuuAdTsdT 3550 1711 antis uAAAuAUCUUcAAGUuAcAdTsdT 35501712 AD-22127-b1 sense GuAAcuuGAAGAuAuuuAudTsdT 3551 1713 antisAuAAAuAUCUUcAAGUuACdTsdT 3551 1714 AD-22128-b1 senseuAAcuuGAAGAuAuuuAuudTsdT 3552 1715 antis AAuAAAuAUCUUcAAGUuAdTsdT 35521716 AD-22129-b1 sense AAcuuGAAGAuAuuuAuucdTsdT 3553 1717 antisGAAuAAAuAUCUUcAAGUUdTsdT 3553 1718 AD-22130-b1 senseAcuuGAAGAuAuuuAuucudTsdT 3554 1719 antis AGAAuAAAuAUCUUcAAGUdTsdT 35541720 AD-22131-b1 sense cuuGAAGAuAuuuAuucuGdTsdT 3555 1721 antiscAGAAuAAAuAUCUUcAAGdTsdT 3555 1722 AD-22132-b1 senseuuGAAGAuAuuuAuucuGGdTsdT 3556 1723 antis CcAGAAuAAAuAUCUUcAAdTsdT 35561724 AD-22133-b1 sense uGAAGAuAuuuAuucuGGGdTsdT 3557 1725 antisCCcAGAAuAAAuAUCUUcAdTsdT 3557 1726 AD-22134-b1 senseGAAGAuAuuuAuucuGGGudTsdT 3558 1727 antis ACCcAGAAuAAAuAUCUUCdTsdT 35581728  *Target: 5′ nutlcoetide of target sequence in human PCSK9 gene,access # NM_174936 U, C, A, G: corresponding ribonucleotide; dT:deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide;Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1corresponds to DFT difluorotoluyl ribo(or deoxyribo) nucleotide; wherenucleotides are written in sequence, they are connected by3′-5′ phosphodiester groups; nucleotides with interjected “s” areconnected by 3′-O-5′-O phosphorothiodiester groups; unless denoted byprefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-mostnucleotide

TABLE 9 Sequences of XBP-1 dsRNAs*Target refers to target gene and location of target sequence. NM_001004210is the gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta(rhesus monkey) XBP-1. SEQ ID SEQ ID Target* NO sense (5′-3′) NOantisense (5′-3′) NM_001004210 1729 CCCAGCUGAUUAGUGUCUA 1753UAGACACUAAUCAGCUGGG 1128-1146 NM_001004210 1730 CCAGCUGAUUAGUGUCUAA 1754UUAGACACUAAUCAGCUGG 1129-1147 NM_001004210 1731 CUCCCAGAGGUCUACCCAG 1755CUGGGUAGACCUCUGGGAG 677-695 NM_001004210 1732 GAUCACCCUGAAUUCAUUG 1756CAAUGAAUUCAGGGUGAUC 893-911 NM_001004210 1733 UCACCCUGAAUUCAUUGUC 1757GACAAUGAAUUCAGGGUGA 895-913 NM_001004210 1734 CCCCAGCUGAUUAGUGUCU 1758AGACACUAAUCAGCUGGGG 1127-1145 NM_001004210 1735 AUCACCCUGAAUUCAUUGU 1759ACAAUGAAUUCAGGGUGAU 894-912 NM_001004210 1736 CAUUUAUUUAAAACUACCC 1760GGGUAGUUUUAAAUAAAUG 1760-1778 NM_001004210 1737 ACUGAAAAACAGAGUAGCA 1761UGCUACUCUGUUUUUCAGU 215-233 NM_001004210 1738 CCAUUUAUUUAAAACUACC 1762GGUAGUUUUAAAUAAAUGG 1759-1777 NM_001004210 1739 UUGAGAACCAGGAGUUAAG 1763CUUAACUCCUGGUUCUCAA 367-385 NM_001004210 1740 CACCCUGAAUUCAUUGUCU 1764AGACAAUGAAUUCAGGGUG 896-914 NM_001004210 1741 AACUGAAAAACAGAGUAGC 1765GCUACUCUGUUUUUCAGUU 214-232 NM_001004210 1742 CUGAAAAACAGAGUAGCAG 1766CUGCUACUCUGUUUUUCAG 216-234 XM_001103095 1743 AGAAAAUCAGCUUUUACGA 1767UCGUAAAAGCUGAUUUUCU 387-405 XM_001103095 1744 UCCCCAGCUGAUUAGUGUC 1768GACACUAAUCAGCUGGGGA 1151-1169 XM_001103095 1745 UACUUAUUAUGUAAGGGUC 1769GACCCUUACAUAAUAAGUA 1466-1484 XM_001103095 1746 UAUCUUAAAAGGGUGGUAG 1770CUACCACCCUUUUAAGAUA 1435-1453 XM_001103095 1747 CCAUGGAUUCUGGCGGUAU 1771AUACCGCCAGAAUCCAUGG 577-595 XM_001103095 1748 UUAAUGAACUAAUUCGUUU 1772AAACGANUUAGUUCAUUAA 790-808 XM_001103095 1749 AGGGUCAUUAGACAAAUGU 1773ACAUUUGUCUAAUGACCCU 1479-1497 XM_001103095 1750 UGAACUAAUUCGUUUUGAC 1774GUCAAAACGAAUUAGUUCA 794-812 XM_001103095 1751 UUCCCCAGCUGAUUAGUGU 1775ACACUAAUCAGCUGGGGAA 1150-1168 XM_001103095 1752 UAUGUAAGGGUCAUUAGAC 1776GUCUAAUGACCCUUACAUA 1473-1491

TABLE 10 Target gene name and target sequence location fordsRNA targeting XBP-1 *Target refers to target gene and location oftarget sequence. NM_001004210 is the gene for ratXBP-1. XM_001103095 is the sequence for Macacamulatta (rhesus monkey) XBP-1. Duplex  Target gene and location #of target sequence D18027 NM_001004210_1128-1146 D18028NM_001004210_1129-1147 D18029 NM_001004210_677-695 D18030NM_001004210_893-911 D18031 NM_001004210_895-913 D18032NM_001004210_1127-1145 D18033 NM_001004210_894-912 D18034NM_001004210_1760-1778 AD18035 NM_001004210_215-233 AD18036NM_001004210_1759-1777 AD18037 NM_001004210_367-385 AD18038NM_001004210_896-914 AD18039 NM_001004210_214-232 AD18040NM_001004210_216-234 AD18041 XM_001103095_387-405 AD18042XM_001103095_1151-1169 AD18043 XM_001103095_1466-1484 AD18044XM_001103095_1435-1453 AD18045 XM_001103095_577-595 AD18046XM_001103095_790-808 AD18047 XM_001103095_1479-1497 AD18048XM_001103095_794-812 AD18049 XM_001103095_1150-1168 AD18050XM_001103095_1473-1491

TABLE 11Sequences of dsRNA targeting XBP-1, with Endolight chemistry modificationsU, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; where nucleotides are written insequence, they are connected by 3′-5′ phosphodiester groups; nucleotides withinterjected ″s″ are connected by 3′-O-5′-O phosphorothiodiester groups.SEQ SEQ ID ID Duplex # NO Sense (5′-3′) NO Antisense (5′-3′) AD180274166 cccAGcuGAuuAGuGucuAdTsdT 1800 uAGAcACuAAUcAGCUGGGdTsdT AD18028 1777ccAGcuGAuuAGuGucuAAdTsdT 1801 UuAGAcACuAAUcAGCUGGdTsdT AD18029 1778cucccAGAGGucuAcccAGdTsdT 1802 CUGGGuAGACCUCUGGGAGdTsdT AD18030 1779GAucAcccuGAAuucAuuGdTsdT 1803 cAAUGAAUUcAGGGUGAUCdTsdT AD18031 1780ucAcccuGAAuucAuuGucdTsdT 1804 GAcAAUGAAUUcAGGGUGAdTsdT AD18032 1781ccccAGcuGAuuAGuGucudTsdT 1805 AGAcACuAAUcAGCUGGGGdTsdT AD18033 1782AucAcccuGAAuucAuuGudTsdT 1806 AcAAUGAAUUcAGGGUGAUdTsdT AD18034 1783cAuuuAuuuAAAAcuAcccdTsdT 1807 GGGuAGUUUuAAAuAAAUGdTsdT AD18035 1784AcuGAAAAAcAGAGuAGcAdTsdT 1808 UGCuACUCUGUUUUUcAGUdTsdT AD18036 1785ccAuuuAuuuAAAAcuAccdTsdT 1809 GGuAGUUUuAAAuAAAUGGdTsdT AD18037 1786uuGAGAAccAGGAGuuAAGdTsdT 1810 CUuAACUCCUGGUUCUcAAdTsdT AD18038 1787cAcccuGAAuucAuuGucudTsdT 1811 AGAcAAUGAAUUcAGGGUGdTsdT AD18039 1788AAcuGAAAAAcAGAGuAGcdTsdT 1812 GCuACUCUGUUUUUcAGUUdTsdT AD18040 1789cuGAAAAAcAGAGuAGcAGdTsdT 1813 CUGCuACUCUGUUUUUcAGdTsdT AD18041 1790AGAAAAucAGcuuuuAcGAdTsdT 1814 UCGuAAAAGCUGAUUUUCUdTsdT AD18042 1791uccccAGcuGAuuAGuGucdTsdT 1815 GAcACuAAUcAGCUGGGGAdTsdT AD18043 1792uAcuuAuuAuGuAAGGGucdTsdT 1816 GACCCUuAcAuAAuAAGuAdTsdT AD18044 1793uAucuuAAAAGGGuGGuAGdTsdT 1817 CuACcACCCUUUuAAGAuAdTsdT AD18045 1794ccAuGGAuucuGGcGGuAudTsdT 1818 AuACCGCcAGAAUCcAUGGdTsdT AD18046 1795uuAAuGAAcuAAuucGuuudTsdT 1819 AAACGAAUuAGUUcAUuAAdTsdT AD18047 1796AGGGucAuuAGAcAAAuGudTsdT 1820 AcAUUUGUCuAAUGACCCUdTsdT AD18048 1797uGAAcuAAuucGuuuuGAcdTsdT 1821 GUcAAAACGAAUuAGUUcAdTsdT AD18049 1798uuccccAGcuGAuuAGuGudTsdT 1822 AcACuAAUcAGCUGGGGAAdTsdT AD18050 1799uAuGuAAGGGucAuuAGAcdTsdT 1823 GUCuAAUGACCCUuAcAuAdTsdT

TABLE 12Sequences of dsRNA targeting both human and rhesus monkey XBP-1.*Target refers location of target sequence in NM_005080 (human XBP-1 mRNA).Sense and antisense sequences are described with optional dinucleotide (NN)overhangs. SEQ ID SEQ Target* sense (5′-3′) NO antisense (5′-3′) ID NO 100-118 CUGCUUCUGUCGGGGCAGCNN 1824 GCUGCCCCGACAGAAGCAGNN 28 1011-1029GAGCUGGGUAUCUCAAAUCNN 1825 GAUUUGAGAUACCCAGCUCNN 28  101-119UGCUUCUGUCGGGGCAGCCNN 1826 GGCUGCCCCGACAGAAGCANN 28 1012-1030AGCUGGGUAUCUCAAAUCUNN 1827 AGAUUUGAGAUACCCAGCUNN 28 1013-1031GCUGGGUAUCUCAAAUCUGNN 1828 CAGAUUUGAGAUACCCAGCNN 28 1014-1032CUGGGUAUCUCAAAUCUGCNN 1829 GCAGAUUUGAGAUACCCAGNN 28 1015-1033UGGGUAUCUCAAAUCUGCUNN 1830 AGCAGAUUUGAGAUACCCANN 28 1016-1034GGGUAUCUCAAAUCUGCUUNN 1831 AAGCAGAUUUGAGAUACCCNN 28 1017-1035GGUAUCUCAAAUCUGCUUUNN 1832 AAAGCAGAUUUGAGAUACCNN 28 1018-1036GUAUCUCAAAUCUGCUUUCNN 1833 GAAAGCAGAUUUGAGAUACNN 28 1019-1037UAUCUCAAAUCUGCUUUCANN 1834 UGAAAGCAGAUUUGAGAUANN 28 1020-1038AUCUCAAAUCUGCUUUCAUNN 1835 AUGAAAGCAGAUUUGAGAUNN 28 1021-1039UCUCAAAUCUGCUUUCAUCNN 1836 GAUGAAAGCAGAUUUGAGANN 28  102-120GCUUCUGUCGGGGCAGCCCNN 1837 GGGCUGCCCCGACAGAAGCNN 28 1022-1040CUCAAAUCUGCUUUCAUCCNN 1838 GGAUGAAAGCAGAUUUGAGNN 28 1023-1041UCAAAUCUGCUUUCAUCCANN 1839 UGGAUGAAAGCAGAUUUGANN 28 1024-1042CAAAUCUGCUUUCAUCCAGNN 1840 CUGGAUGAAAGCAGAUUUGNN 28 1025-1043AAAUCUGCUUUCAUCCAGCNN 1841 GCUGGAUGAAAGCAGAUUUNN 29 1026-1044AAUCUGCUUUCAUCCAGCCNN 1842 GGCUGGAUGAAAGCAGAUUNN 29 1027-1045AUCUGCUUUCAUCCAGCCANN 1843 UGGCUGGAUGAAAGCAGAUNN 29 1028-1046UCUGCUUUCAUCCAGCCACNN 1844 GUGGCUGGAUGAAAGCAGANN 29 1029-1047CUGCUUUCAUCCAGCCACUNN 1845 AGUGGCUGGAUGAAAGCAGNN 29 1030-1048UGCUUUCAUCCAGCCACUGNN 1846 CAGUGGCUGGAUGAAAGCANN 29 1031-1049GCUUUCAUCCAGCCACUGCNN 1847 GCAGUGGCUGGAUGAAAGCNN 29  103-121CUUCUGUCGGGGCAGCCCGNN 1848 CGGGCUGCCCCGACAGAAGNN 29 1032-1050CUUUCAUCCAGCCACUGCCNN 1849 GGCAGUGGCUGGAUGAAAGNN 29 1033-1051UUUCAUCCAGCCACUGCCCNN 1850 GGGCAGUGGCUGGAUGAAANN 29  104-122UUCUGUCGGGGCAGCCCGCNN 1851 GCGGGCUGCCCCGACAGAANN 29  105-123UCUGUCGGGGCAGCCCGCCNN 1852 GGCGGGCUGCCCCGACAGANN 29 1056-1074CCAUCUUCCUGCCUACUGGNN 1853 CCAGUAGGCAGGAAGAUGGNN 29 1057-1075CAUCUUCCUGCCUACUGGANN 1854 UCCAGUAGGCAGGAAGAUGNN 29 1058-1076AUCUUCCUGCCUACUGGAUNN 1855 AUCCAGUAGGCAGGAAGAUNN 29 1059-1077UCUUCCUGCCUACUGGAUGNN 1856 CAUCCAGUAGGCAGGAAGANN 29 1060-1078CUUCCUGCCUACUGGAUGCNN 1857 GCAUCCAGUAGGCAGGAAGNN 29 1061-1079UUCCUGCCUACUGGAUGCUNN 1858 AGCAUCCAGUAGGCAGGAANN 29  106-124CUGUCGGGGCAGCCCGCCUNN 1859 AGGCGGGCUGCCCCGACAGNN 29 1062-1080UCCUGCCUACUGGAUGCUUNN 1860 AAGCAUCCAGUAGGCAGGANN 29 1063-1081CCUGCCUACUGGAUGCUUANN 1861 UAAGCAUCCAGUAGGCAGGNN 29 1064-1082CUGCCUACUGGAUGCUUACNN 1862 GUAAGCAUCCAGUAGGCAGNN 29 1065-1083UGCCUACUGGAUGCUUACANN 1863 UGUAAGCAUCCAGUAGGCANN 29 1066-1084GCCUACUGGAUGCUUACAGNN 1864 CUGUAAGCAUCCAGUAGGCNN 29 1067-1085CCUACUGGAUGCUUACAGUNN 1865 ACUGUAAGCAUCCAGUAGGNN 29 1068-1086CUACUGGAUGCUUACAGUGNN 1866 CACUGUAAGCAUCCAGUAGNN 29 1069-1087UACUGGAUGCUUACAGUGANN 1867 UCACUGUAAGCAUCCAGUANN 29 1070-1088ACUGGAUGCUUACAGUGACNN 1868 GUCACUGUAAGCAUCCAGUNN 29 1071-1089CUGGAUGCUUACAGUGACUNN 1869 AGUCACUGUAAGCAUCCAGNN 29  107-125UGUCGGGGCAGCCCGCCUCNN 1870 GAGGCGGGCUGCCCCGACANN 29 1072-1090UGGAUGCUUACAGUGACUGNN 1871 CAGUCACUGUAAGCAUCCANN 29 1073-1091GGAUGCUUACAGUGACUGUNN 1872 ACAGUCACUGUAAGCAUCCNN 29 1074-1092GAUGCUUACAGUGACUGUGNN 1873 CACAGUCACUGUAAGCAUCNN 29 1075-1093AUGCUUACAGUGACUGUGGNN 1874 CCACAGUCACUGUAAGCAUNN 29 1076-1094UGCUUACAGUGACUGUGGANN 1875 UCCACAGUCACUGUAAGCANN 29 1077-1095GCUUACAGUGACUGUGGAUNN 1876 AUCCACAGUCACUGUAAGCNN 29 1078-1096CUUACAGUGACUGUGGAUANN 1877 UAUCCACAGUCACUGUAAGNN 29  108-126GUCGGGGCAGCCCGCCUCCNN 1878 GGAGGCGGGCUGCCCCGACNN 29  109-127UCGGGGCAGCCCGCCUCCGNN 1879 CGGAGGCGGGCUGCCCCGANN 29  110-128CGGGGCAGCCCGCCUCCGCNN 1880 GCGGAGGCGGGCUGCCCCGNN 29  111-129GGGGCAGCCCGCCUCCGCCNN 1881 GGCGGAGGCGGGCUGCCCCNN 29 1116-1134UUCAGUGACAUGUCCUCUCNN 1882 GAGAGGACAUGUCACUGAANN 29  112-130GGGCAGCCCGCCUCCGCCGNN 1883 CGGCGGAGGCGGGCUGCCCNN 29  113-131GGCAGCCCGCCUCCGCCGCNN 1884 GCGGCGGAGGCGGGCUGCCNN 29 1136-1154GCUUGGUGUAAACCAUUCUNN 1885 AGAAUGGUUUACACCAAGCNN 29 1137-1155CUUGGUGUAAACCAUUCUUNN 1886 AAGAAUGGUUUACACCAAGNN 29 1138-1156UUGGUGUAAACCAUUCUUGNN 1887 CAAGAAUGGUUUACACCAANN 29 1139-1157UGGUGUAAACCAUUCUUGGNN 1888 CCAAGAAUGGUUUACACCANN 29 1140-1158GGUGUAAACCAUUCUUGGGNN 1889 CCCAAGAAUGGUUUACACCNN 29 1141-1159GUGUAAACCAUUCUUGGGANN 1890 UCCCAAGAAUGGUUUACACNN 29  114-132GCAGCCCGCCUCCGCCGCCNN 1891 GGCGGCGGAGGCGGGCUGCNN 29 1142-1160UGUAAACCAUUCUUGGGAGNN 1892 CUCCCAAGAAUGGUUUACANN 29 1143-1161GUAAACCAUUCUUGGGAGGNN 1893 CCUCCCAAGAAUGGUUUACNN 29 1144-1162UAAACCAUUCUUGGGAGGANN 1894 UCCUCCCAAGAAUGGUUUANN 29 1145-1163AAACCAUUCUUGGGAGGACNN 1895 GUCCUCCCAAGAAUGGUUUNN 29 1146-1164AACCAUUCUUGGGAGGACANN 1896 UGUCCUCCCAAGAAUGGUUNN 29 1147-1165ACCAUUCUUGGGAGGACACNN 1897 GUGUCCUCCCAAGAAUGGUNN 29 1148-1166CCAUUCUUGGGAGGACACUNN 1898 AGUGUCCUCCCAAGAAUGGNN 29 1149-1167CAUUCUUGGGAGGACACUUNN 1899 AAGUGUCCUCCCAAGAAUGNN 29 1150-1168AUUCUUGGGAGGACACUUUNN 1900 AAAGUGUCCUCCCAAGAAUNN 29 1151-1169UUCUUGGGAGGACACUUUUNN 1901 AAAAGUGUCCUCCCAAGAANN 29  115-133CAGCCCGCCUCCGCCGCCGNN 1902 CGGCGGCGGAGGCGGGCUGNN 29 1152-1170UCUUGGGAGGACACUUUUGNN 1903 CAAAAGUGUCCUCCCAAGANN 29 1153-1171CUUGGGAGGACACUUUUGCNN 1904 GCAAAAGUGUCCUCCCAAGNN 29 1154-1172UUGGGAGGACACUUUUGCCNN 1905 GGCAAAAGUGUCCUCCCAANN 29 1155-1173UGGGAGGACACUUUUGCCANN 1906 UGGCAAAAGUGUCCUCCCANN 29 1156-1174GGGAGGACACUUUUGCCAANN 1907 UUGGCAAAAGUGUCCUCCCNN 29 1157-1175GGAGGACACUUUUGCCAAUNN 1908 AUUGGCAAAAGUGUCCUCCNN 29 1158-1176GAGGACACUUUUGCCAAUGNN 1909 CAUUGGCAAAAGUGUCCUCNN 29 1159-1177AGGACACUUUUGCCAAUGANN 1910 UCAUUGGCAAAAGUGUCCUNN 29 1160-1178GGACACUUUUGCCAAUGAANN 1911 UUCAUUGGCAAAAGUGUCCNN 29 1161-1179GACACUUUUGCCAAUGAACNN 1912 GUUCAUUGGCAAAAGUGUCNN 29  116-134AGCCCGCCUCCGCCGCCGGNN 1913 CCGGCGGCGGAGGCGGGCUNN 29 1162-1180ACACUUUUGCCAAUGAACUNN 1914 AGUUCAUUGGCAAAAGUGUNN 29  117-135GCCCGCCUCCGCCGCCGGANN 1915 UCCGGCGGCGGAGGCGGGCNN 29  118-136CCCGCCUCCGCCGCCGGAGNN 1916 CUCCGGCGGCGGAGGCGGGNN 29 1182-1200UUUCCCCAGCUGAUUAGUGNN 1917 CACUAAUCAGCUGGGGAAANN 29 1183-1201UUCCCCAGCUGAUUAGUGUNN 1918 ACACUAAUCAGCUGGGGAANN 29 1184-1202UCCCCAGCUGAUUAGUGUCNN 1919 GACACUAAUCAGCUGGGGANN 29 1185-1203CCCCAGCUGAUUAGUGUCUNN 1920 AGACACUAAUCAGCUGGGGNN 29 1186-1204CCCAGCUGAUUAGUGUCUANN 1921 UAGACACUAAUCAGCUGGGNN 29 1187-1205CCAGCUGAUUAGUGUCUAANN 1922 UUAGACACUAAUCAGCUGGNN 29 1188-1206CAGCUGAUUAGUGUCUAAGNN 1923 CUUAGACACUAAUCAGCUGNN 29 1189-1207AGCUGAUUAGUGUCUAAGGNN 1924 CCUUAGACACUAAUCAGCUNN 29 1190-1208GCUGAUUAGUGUCUAAGGANN 1925 UCCUUAGACACUAAUCAGCNN 29 1191-1209CUGAUUAGUGUCUAAGGAANN 1926 UUCCUUAGACACUAAUCAGNN 29  119-137CCGCCUCCGCCGCCGGAGCNN 1927 GCUCCGGCGGCGGAGGCGGNN 29 1192-1210UGAUUAGUGUCUAAGGAAUNN 1928 AUUCCUUAGACACUAAUCANN 29 1193-1211GAUUAGUGUCUAAGGAAUGNN 1929 CAUUCCUUAGACACUAAUCNN 29 1194-1212AUUAGUGUCUAAGGAAUGANN 1930 UCAUUCCUUAGACACUAAUNN 29 1195-1213UUAGUGUCUAAGGAAUGAUNN 1931 AUCAUUCCUUAGACACUAANN 29 1196-1214UAGUGUCUAAGGAAUGAUCNN 1932 GAUCAUUCCUUAGACACUANN 29 1197-1215AGUGUCUAAGGAAUGAUCCNN 1933 GGAUCAUUCCUUAGACACUNN 29 1198-1216GUGUCUAAGGAAUGAUCCANN 1934 UGGAUCAUUCCUUAGACACNN 29  120-138CGCCUCCGCCGCCGGAGCCNN 1935 GGCUCCGGCGGCGGAGGCGNN 29  121-139GCCUCCGCCGCCGGAGCCCNN 1936 GGGCUCCGGCGGCGGAGGCNN 29 1218-1236UACUGUUGCCCUUUUCCUUNN 1937 AAGGAAAAGGGCAACAGUANN 29 1219-1237ACUGUUGCCCUUUUCCUUGNN 1938 CAAGGAAAAGGGCAACAGUNN 29 1220-1238CUGUUGCCCUUUUCCUUGANN 1939 UCAAGGAAAAGGGCAACAGNN 29 1221-1239UGUUGCCCUUUUCCUUGACNN 1940 GUCAAGGAAAAGGGCAACANN 29  122-140CCUCCGCCGCCGGAGCCCCNN 1941 GGGGCUCCGGCGGCGGAGGNN 30 1222-1240GUUGCCCUUUUCCUUGACUNN 1942 AGUCAAGGAAAAGGGCAACNN 30 1223-1241UUGCCCUUUUCCUUGACUANN 1943 UAGUCAAGGAAAAGGGCAANN 30 1224-1242UGCCCUUUUCCUUGACUAUNN 1944 AUAGUCAAGGAAAAGGGCANN 30 1225-1243GCCCUUUUCCUUGACUAUUNN 1945 AAUAGUCAAGGAAAAGGGCNN 30 1226-1244CCCUUUUCCUUGACUAUUANN 1946 UAAUAGUCAAGGAAAAGGGNN 30 1227-1245CCUUUUCCUUGACUAUUACNN 1947 GUAAUAGUCAAGGAAAAGGNN 30 1228-1246CUUUUCCUUGACUAUUACANN 1948 UGUAAUAGUCAAGGAAAAGNN 30 1229-1247UUUUCCUUGACUAUUACACNN 1949 GUGUAAUAGUCAAGGAAAANN 30 1230-1248UUUCCUUGACUAUUACACUNN 1950 AGUGUAAUAGUCAAGGAAANN 30 1231-1249UUCCUUGACUAUUACACUGNN 1951 CAGUGUAAUAGUCAAGGAANN 30  123-141CUCCGCCGCCGGAGCCCCGNN 1952 CGGGGCUCCGGCGGCGGAGNN 30 1232-1250UCCUUGACUAUUACACUGCNN 1953 GCAGUGUAAUAGUCAAGGANN 30 1233-1251CCUUGACUAUUACACUGCCNN 1954 GGCAGUGUAAUAGUCAAGGNN 30 1234-1252CUUGACUAUUACACUGCCUNN 1955 AGGCAGUGUAAUAGUCAAGNN 30 1235-1253UUGACUAUUACACUGCCUGNN 1956 CAGGCAGUGUAAUAGUCAANN 30 1236-1254UGACUAUUACACUGCCUGGNN 1957 CCAGGCAGUGUAAUAGUCANN 30 1237-1255GACUAUUACACUGCCUGGANN 1958 UCCAGGCAGUGUAAUAGUCNN 30 1238-1256ACUAUUACACUGCCUGGAGNN 1959 CUCCAGGCAGUGUAAUAGUNN 30 1239-1257CUAUUACACUGCCUGGAGGNN 1960 CCUCCAGGCAGUGUAAUAGNN 30 1240-1258UAUUACACUGCCUGGAGGANN 1961 UCCUCCAGGCAGUGUAAUANN 30 1241-1259AUUACACUGCCUGGAGGAUNN 1962 AUCCUCCAGGCAGUGUAAUNN 30  124-142UCCGCCGCCGGAGCCCCGGNN 1963 CCGGGGCUCCGGCGGCGGANN 30 1242-1260UUACACUGCCUGGAGGAUANN 1964 UAUCCUCCAGGCAGUGUAANN 30 1243-1261UACACUGCCUGGAGGAUAGNN 1965 CUAUCCUCCAGGCAGUGUANN 30 1244-1262ACACUGCCUGGAGGAUAGCNN 1966 GCUAUCCUCCAGGCAGUGUNN 30 1245-1263CACUGCCUGGAGGAUAGCANN 1967 UGCUAUCCUCCAGGCAGUGNN 30 1246-1264ACUGCCUGGAGGAUAGCAGNN 1968 CUGCUAUCCUCCAGGCAGUNN 30  125-143CCGCCGCCGGAGCCCCGGCNN 1969 GCCGGGGCTCCGGCGGCGGNN 30  126-144CGCCGCCGGAGCCCCGGCCNN 1970 GGCCGGGGCTCCGGCGGCGNN 30  127-145GCCGCCGGAGCCCCGGCCGNN 1971 CGGCCGGGGCTCCGGCGGCNN 30 1280-1298CUUCAUUCAAAAAGCCAAANN 1972 UUUGGCUUUUUGAAUGAAGNN 30 1281-1299UUCAUUCAAAAAGCCAAAANN 1973 UUUUGGCUUUUUGAAUGAANN 30  128-146CCGCCGGAGCCCCGGCCGGNN 1974 CCGGCCGGGGCTCCGGCGGNN 30 1282-1300UCAUUCAAAAAGCCAAAAUNN 1975 AUUUUGGCUUUUUGAAUGANN 30 1283-1301CAUUCAAAAAGCCAAAAUANN 1976 UAUUUUGGCUUUUUGAAUGNN 30 1284-1302AUUCAAAAAGCCAAAAUAGNN 1977 CUAUUUUGGCUUUUUGAAUNN 30 1285-1303UUCAAAAAGCCAAAAUAGANN 1978 UCUAUUUUGGCUUUUUGAANN 30 1286-1304UCAAAAAGCCAAAAUAGAGNN 1979 CUCUAUUUUGGCUUUUUGANN 30 1287-1305CAAAAAGCCAAAAUAGAGANN 1980 UCUCUAUUUUGGCUUUUUGNN 30 1288-1306AAAAAGCCAAAAUAGAGAGNN 1981 CUCUCUAUUUUGGCUUUUUNN 30 1289-1307AAAAGCCAAAAUAGAGAGUNN 1982 ACUCUCUAUUUUGGCUUUUNN 30 1290-1308AAAGCCAAAAUAGAGAGUANN 1983 UACUCUCUAUUUUGGCUUUNN 30  129-147CGCCGGAGCCCCGGCCGGCNN 1984 GCCGGCCGGGGCTCCGGCGNN 30  130-148GCCGGAGCCCCGGCCGGCCNN 1985 GGCCGGCCGGGGCTCCGGCNN 30 1310-1328ACAGUCCUAGAGAAUUCCUNN 1986 AGGAAUUCUCUAGGACUGUNN 30  131-149CCGGAGCCCCGGCCGGCCANN 1987 TGGCCGGCCGGGGCTCCGGNN 30  132-150CGGAGCCCCGGCCGGCCAGNN 1988 CTGGCCGGCCGGGGCTCCGNN 30 1330-1348UAUUUGUUCAGAUCUCAUANN 1989 UAUGAGAUCUGAACAAAUANN 30 1331-1349AUUUGUUCAGAUCUCAUAGNN 1990 CUAUGAGAUCUGAACAAAUNN 30  133-151GGAGCCCCGGCCGGCCAGGNN 1991 CCTGGCCGGCCGGGGCTCCNN 30 1332-1350UUUGUUCAGAUCUCAUAGANN 1992 UCUAUGAGAUCUGAACAAANN 30 1333-1351UUGUUCAGAUCUCAUAGAUNN 1993 AUCUAUGAGAUCUGAACAANN 30 1334-1352UGUUCAGAUCUCAUAGAUGNN 1994 CAUCUAUGAGAUCUGAACANN 30 1335-1353GUUCAGAUCUCAUAGAUGANN 1995 UCAUCUAUGAGAUCUGAACNN 30  134-152GAGCCCCGGCCGGCCAGGCNN 1996 GCCTGGCCGGCCGGGGCTCNN 30  135-153AGCCCCGGCCGGCCAGGCCNN 1997 GGCCTGGCCGGCCGGGGCTNN 30  136-154GCCCCGGCCGGCCAGGCCCNN 1998 GGGCCTGGCCGGCCGGGGCNN 30 1365-1383UGUCUUUUGACAUCCAGCANN 1999 UGCUGGAUGUCAAAAGACANN 30 1366-1384GUCUUUUGACAUCCAGCAGNN 2000 CUGCUGGAUGUCAAAAGACNN 30 1367-1385UCUUUUGACAUCCAGCAGUNN 2001 ACUGCUGGAUGUCAAAAGANN 30 1368-1386CUUUUGACAUCCAGCAGUCNN 2002 GACUGCUGGAUGUCAAAAGNN 30 1369-1387UUUUGACAUCCAGCAGUCCNN 2003 GGACUGCUGGAUGUCAAAANN 30 1370-1388UUUGACAUCCAGCAGUCCANN 2004 UGGACUGCUGGAUGUCAAANN 30 1371-1389UUGACAUCCAGCAGUCCAANN 2005 UUGGACUGCUGGAUGUCAANN 30  137-155CCCCGGCCGGCCAGGCCCUNN 2006 AGGGCCUGGCCGGCCGGGGNN 30  138-156CCCGGCCGGCCAGGCCCUGNN 2007 CAGGGCCUGGCCGGCCGGGNN 30 1391-1409GUAUUGAGACAUAUUACUGNN 2008 CAGUAAUAUGUCUCAAUACNN 30  139-157CCGGCCGGCCAGGCCCUGCNN 2009 GCAGGGCCUGGCCGGCCGGNN 30  140-158CGGCCGGCCAGGCCCUGCCNN 2010 GGCAGGGCCUGGCCGGCCGNN 30  141-159GGCCGGCCAGGCCCUGCCGNN 2011 CGGCAGGGCCUGGCCGGCCNN 30 1414-1432UAAGAAAUAUUACUAUAAUNN 2012 AUUAUAGUAAUAUUUCUUANN 30 1415-1433AAGAAAUAUUACUAUAAUUNN 2013 AAUUAUAGUAAUAUUUCUUNN 30 1416-1434AGAAAUAUUACUAUAAUUGNN 2014 CAAUUAUAGUAAUAUUUCUNN 30 1417-1435GAAAUAUUACUAUAAUUGANN 2015 UCAAUUAUAGUAAUAUUUCNN 30 1418-1436AAAUAUUACUAUAAUUGAGNN 2016 CUCAAUUAUAGUAAUAUUUNN 30 1419-1437AAUAUUACUAUAAUUGAGANN 2017 UCUCAAUUAUAGUAAUAUUNN 30 1420-1438AUAUUACUAUAAUUGAGAANN 2018 UUCUCAAUUAUAGUAAUAUNN 30 1421-1439UAUUACUAUAAUUGAGAACNN 2019 GUUCUCAAUUAUAGUAAUANN 30  142-160GCCGGCCAGGCCCUGCCGCNN 2020 GCGGCAGGGCCUGGCCGGCNN 30 1422-1440AUUACUAUAAUUGAGAACUNN 2021 AGUUCUCAAUUAUAGUAAUNN 30 1423-1441UUACUAUAAUUGAGAACUANN 2022 UAGUUCUCAAUUAUAGUAANN 30 1424-1442UACUAUAAUUGAGAACUACNN 2023 GUAGUUCUCAAUUAUAGUANN 30 1425-1443ACUAUAAUUGAGAACUACANN 2024 UGUAGUUCUCAAUUAUAGUNN 30 1426-1444CUAUAAUUGAGAACUACAGNN 2025 CUGUAGUUCUCAAUUAUAGNN 30 1427-1445UAUAAUUGAGAACUACAGCNN 2026 GCUGUAGUUCUCAAUUAUANN 30 1428-1446AUAAUUGAGAACUACAGCUNN 2027 AGCUGUAGUUCUCAAUUAUNN 30 1429-1447UAAUUGAGAACUACAGCUUNN 2028 AAGCUGUAGUUCUCAAUUANN 30 1430-1448AAUUGAGAACUACAGCUUUNN 2029 AAAGCUGUAGUUCUCAAUUNN 30 1431-1449AUUGAGAACUACAGCUUUUNN 2030 AAAAGCUGUAGUUCUCAAUNN 30  143-161CCGGCCAGGCCCUGCCGCUNN 2031 AGCGGCAGGGCCUGGCCGGNN 30 1432-1450UUGAGAACUACAGCUUUUANN 2032 UAAAAGCUGUAGUUCUCAANN 30 1433-1451UGAGAACUACAGCUUUUAANN 2033 UUAAAAGCUGUAGUUCUCANN 30 1434-1452GAGAACUACAGCUUUUAAGNN 2034 CUUAAAAGCUGUAGUUCUCNN 30 1435-1453AGAACUACAGCUUUUAAGANN 2035 UCUUAAAAGCUGUAGUUCUNN 30 1436-1454GAACUACAGCUUUUAAGAUNN 2036 AUCUUAAAAGCUGUAGUUCNN 30 1437-1455AACUACAGCUUUUAAGAUUNN 2037 AAUCUUAAAAGCUGUAGUUNN 30 1438-1456ACUACAGCUUUUAAGAUUGNN 2038 CAAUCUUAAAAGCUGUAGUNN 30 1439-1457CUACAGCUUUUAAGAUUGUNN 2039 ACAAUCUUAAAAGCUGUAGNN 30 1440-1458UACAGCUUUUAAGAUUGUANN 2040 UACAAUCUUAAAAGCUGUANN 30 1441-1459ACAGCUUUUAAGAUUGUACNN 2041 GUACAAUCUUAAAAGCUGUNN 31  144-162CGGCCAGGCCCUGCCGCUCNN 2042 GAGCGGCAGGGCCUGGCCGNN 31 1442-1460CAGCUUUUAAGAUUGUACUNN 2043 AGUACAAUCUUAAAAGCUGNN 31 1443-1461AGCUUUUAAGAUUGUACUUNN 2044 AAGUACAAUCUUAAAAGCUNN 31 1444-1462GCUUUUAAGAUUGUACUUUNN 2045 AAAGUACAAUCUUAAAAGCNN 31 1445-1463CUUUUAAGAUUGUACUUUUNN 2046 AAAAGUACAAUCUUAAAAGNN 31 1446-1464UUUUAAGAUUGUACUUUUANN 2047 UAAAAGUACAAUCUUAAAANN 31 1447-1465UUUAAGAUUGUACUUUUAUNN 2048 AUAAAAGUACAAUCUUAAANN 31 1448-1466UUAAGAUUGUACUUUUAUCNN 2049 GAUAAAAGUACAAUCUUAANN 31 1449-1467UAAGAUUGUACUUUUAUCUNN 2050 AGAUAAAAGUACAAUCUUANN 31 1450-1468AAGAUUGUACUUUUAUCUUNN 2051 AAGAUAAAAGUACAAUCUUNN 31 1451-1469AGAUUGUACUUUUAUCUUANN 2052 UAAGAUAAAAGUACAAUCUNN 31  145-163GGCCAGGCCCUGCCGCUCANN 2053 UGAGCGGCAGGGCCUGGCCNN 31 1452-1470GAUUGUACUUUUAUCUUAANN 2054 UUAAGAUAAAAGUACAAUCNN 31 1453-1471AUUGUACUUUUAUCUUAAANN 2055 UUUAAGAUAAAAGUACAAUNN 31 1454-1472UUGUACUUUUAUCUUAAAANN 2056 UUUUAAGAUAAAAGUACAANN 31 1455-1473UGUACUUUUAUCUUAAAAGNN 2057 CUUUUAAGAUAAAAGUACANN 31 1456-1474GUACUUUUAUCUUAAAAGGNN 2058 CCUUUUAAGAUAAAAGUACNN 31 1457-1475UACUUUUAUCUUAAAAGGGNN 2059 CCCUUUUAAGAUAAAAGUANN 31 1458-1476ACUUUUAUCUUAAAAGGGUNN 2060 ACCCUUUUAAGAUAAAAGUNN 31 1459-1477CUUUUAUCUUAAAAGGGUGNN 2061 CACCCUUUUAAGAUAAAAGNN 31 1460-1478UUUUAUCUUAAAAGGGUGGNN 2062 CCACCCUUUUAAGAUAAAANN 31 1461-1479UUUAUCUUAAAAGGGUGGUNN 2063 ACCACCCUUUUAAGAUAAANN 31  146-164GCCAGGCCCUGCCGCUCAUNN 2064 AUGAGCGGCAGGGCCUGGCNN 31 1462-1480UUAUCUUAAAAGGGUGGUANN 2065 UACCACCCUUUUAAGAUAANN 31 1463-1481UAUCUUAAAAGGGUGGUAGNN 2066 CUACCACCCUUUUAAGAUANN 31 1464-1482AUCUUAAAAGGGUGGUAGUNN 2067 ACUACCACCCUUUUAAGAUNN 31 1465-1483UCUUAAAAGGGUGGUAGUUNN 2068 AACUACCACCCUUUUAAGANN 31 1466-1484CUUAAAAGGGUGGUAGUUUNN 2069 AAACUACCACCCUUUUAAGNN 31  147-165CCAGGCCCUGCCGCUCAUGNN 2070 CAUGAGCGGCAGGGCCUGGNN 31  148-166CAGGCCCUGCCGCUCAUGGNN 2071 CCAUGAGCGGCAGGGCCUGNN 31 1486-1504CCCUAAAAUACUUAUUAUGNN 2072 CAUAAUAAGUAUUUUAGGGNN 31 1487-1505CCUAAAAUACUUAUUAUGUNN 2073 ACAUAAUAAGUAUUUUAGGNN 31 1488-1506CUAAAAUACUUAUUAUGUANN 2074 UACAUAAUAAGUAUUUUAGNN 31 1489-1507UAAAAUACUUAUUAUGUAANN 2075 UUACAUAAUAAGUAUUUUANN 31 1490-1508AAAAUACUUAUUAUGUAAGNN 2076 CUUACAUAAUAAGUAUUUUNN 31 1491-1509AAAUACUUAUUAUGUAAGGNN 2077 CCUUACAUAAUAAGUAUUUNN 31  149-167AGGCCCUGCCGCUCAUGGUNN 2078 ACCAUGAGCGGCAGGGCCUNN 31 1492-1510AAUACUUAUUAUGUAAGGGNN 2079 CCCUUACAUAAUAAGUAUUNN 31 1493-1511AUACUUAUUAUGUAAGGGUNN 2080 ACCCUUACAUAAUAAGUAUNN 31 1494-1512UACUUAUUAUGUAAGGGUCNN 2081 GACCCUUACAUAAUAAGUANN 31 1495-1513ACUUAUUAUGUAAGGGUCANN 2082 UGACCCUUACAUAAUAAGUNN 31 1496-1514CUUAUUAUGUAAGGGUCAUNN 2083 AUGACCCUUACAUAAUAAGNN 31 1497-1515UUAUUAUGUAAGGGUCAUUNN 2084 AAUGACCCUUACAUAAUAANN 31 1498-1516UAUUAUGUAAGGGUCAUUANN 2085 UAAUGACCCUUACAUAAUANN 31 1499-1517AUUAUGUAAGGGUCAUUAGNN 2086 CUAAUGACCCUUACAUAAUNN 31 1500-1518UUAUGUAAGGGUCAUUAGANN 2087 UCUAAUGACCCUUACAUAANN 31 1501-1519UAUGUAAGGGUCAUUAGACNN 2088 GUCUAAUGACCCUUACAUANN 31  150-168GGCCCUGCCGCUCAUGGUGNN 2089 CACCAUGAGCGGCAGGGCCNN 31 1502-1520AUGUAAGGGUCAUUAGACANN 2090 UGUCUAAUGACCCUUACAUNN 31 1503-1521UGUAAGGGUCAUUAGACAANN 2091 UUGUCUAAUGACCCUUACANN 31 1504-1522GUAAGGGUCAUUAGACAAANN 2092 UUUGUCUAAUGACCCUUACNN 31 1505-1523UAAGGGUCAUUAGACAAAUNN 2093 AUUUGUCUAAUGACCCUUANN 31 1506-1524AAGGGUCAUUAGACAAAUGNN 2094 CAUUUGUCUAAUGACCCUUNN 31 1507-1525AGGGUCAUUAGACAAAUGUNN 2095 ACAUUUGUCUAAUGACCCUNN 31 1508-1526GGGUCAUUAGACAAAUGUCNN 2096 GACAUUUGUCUAAUGACCCNN 31 1509-1527GGUCAUUAGACAAAUGUCUNN 2097 AGACAUUUGUCUAAUGACCNN 31 1510-1528GUCAUUAGACAAAUGUCUUNN 2098 AAGACAUUUGUCUAAUGACNN 31 1511-1529UCAUUAGACAAAUGUCUUGNN 2099 CAAGACAUUUGUCUAAUGANN 31  151-169GCCCUGCCGCUCAUGGUGCNN 2100 GCACCAUGAGCGGCAGGGCNN 31 1512-1530CAUUAGACAAAUGUCUUGANN 2101 UCAAGACAUUUGUCUAAUGNN 31 1513-1531AUUAGACAAAUGUCUUGAANN 2102 UUCAAGACAUUUGUCUAAUNN 31 1514-1532UUAGACAAAUGUCUUGAAGNN 2103 CUUCAAGACAUUUGUCUAANN 31 1515-1533UAGACAAAUGUCUUGAAGUNN 2104 ACUUCAAGACAUUUGUCUANN 31 1516-1534AGACAAAUGUCUUGAAGUANN 2105 UACUUCAAGACAUUUGUCUNN 31 1517-1535GACAAAUGUCUUGAAGUAGNN 2106 CUACUUCAAGACAUUUGUCNN 31 1518-1536ACAAAUGUCUUGAAGUAGANN 2107 UCUACUUCAAGACAUUUGUNN 31  152-170CCCUGCCGCUCAUGGUGCCNN 2108 GGCACCAUGAGCGGCAGGGNN 31  153-171CCUGCCGCUCAUGGUGCCANN 2109 UGGCACCAUGAGCGGCAGGNN 31 1541-1559GAAUUUAUGAAUGGUUCUUNN 2110 AAGAACCAUUCAUAAAUUCNN 31  154-172CUGCCGCUCAUGGUGCCAGNN 2111 CUGGCACCAUGAGCGGCAGNN 31 1542-1560AAUUUAUGAAUGGUUCUUUNN 2112 AAAGAACCAUUCAUAAAUUNN 31 1543-1561AUUUAUGAAUGGUUCUUUANN 2113 UAAAGAACCAUUCAUAAAUNN 31 1544-1562UUUAUGAAUGGUUCUUUAUNN 2114 AUAAAGAACCAUUCAUAAANN 31 1545-1563UUAUGAAUGGUUCUUUAUCNN 2115 GAUAAAGAACCAUUCAUAANN 31 1546-1564UAUGAAUGGUUCUUUAUCANN 2116 UGAUAAAGAACCAUUCAUANN 31 1547-1565AUGAAUGGUUCUUUAUCAUNN 2117 AUGAUAAAGAACCAUUCAUNN 31 1548-1566UGAAUGGUUCUUUAUCAUUNN 2118 AAUGAUAAAGAACCAUUCANN 31 1549-1567GAAUGGUUCUUUAUCAUUUNN 2119 AAAUGAUAAAGAACCAUUCNN 31 1550-1568AAUGGUUCUUUAUCAUUUCNN 2120 GAAAUGAUAAAGAACCAUUNN 31 1551-1569AUGGUUCUUUAUCAUUUCUNN 2121 AGAAAUGAUAAAGAACCAUNN 31  155-173UGCCGCUCAUGGUGCCAGCNN 2122 GCUGGCACCAUGAGCGGCANN 31 1552-1570UGGUUCUUUAUCAUUUCUCNN 2123 GAGAAAUGAUAAAGAACCANN 31 1553-1571GGUUCUUUAUCAUUUCUCUNN 2124 AGAGAAAUGAUAAAGAACCNN 31 1554-1572GUUCUUUAUCAUUUCUCUUNN 2125 AAGAGAAAUGAUAAAGAACNN 31 1555-1573UUCUUUAUCAUUUCUCUUCNN 2126 GAAGAGAAAUGAUAAAGAANN 31 1556-1574UCUUUAUCAUUUCUCUUCCNN 2127 GGAAGAGAAAUGAUAAAGANN 31 1557-1575CUUUAUCAUUUCUCUUCCCNN 2128 GGGAAGAGAAAUGAUAAAGNN 31 1558-1576UUUAUCAUUUCUCUUCCCCNN 2129 GGGGAAGAGAAAUGAUAAANN 31 1559-1577UUAUCAUUUCUCUUCCCCCNN 2130 GGGGGAAGAGAAAUGAUAANN 31 1560-1578UAUCAUUUCUCUUCCCCCUNN 2131 AGGGGGAAGAGAAAUGAUANN 31 1561-1579AUCAUUUCUCUUCCCCCUUNN 2132 AAGGGGGAAGAGAAAUGAUNN 31  156-174GCCGCUCAUGGUGCCAGCCNN 2133 GGCUGGCACCAUGAGCGGCNN 31 1562-1580UCAUUUCUCUUCCCCCUUUNN 2134 AAAGGGGGAAGAGAAAUGANN 31 1563-1581CAUUUCUCUUCCCCCUUUUNN 2135 AAAAGGGGGAAGAGAAAUGNN 31 1564-1582AUUUCUCUUCCCCCUUUUUNN 2136 AAAAAGGGGGAAGAGAAAUNN 31 1565-1583UUUCUCUUCCCCCUUUUUGNN 2137 CAAAAAGGGGGAAGAGAAANN 31 1566-1584UUCUCUUCCCCCUUUUUGGNN 2138 CCAAAAAGGGGGAAGAGAANN 31 1567-1585UCUCUUCCCCCUUUUUGGCNN 2139 GCCAAAAAGGGGGAAGAGANN 31 1568-1586CUCUUCCCCCUUUUUGGCANN 2140 UGCCAAAAAGGGGGAAGAGNN 31 1569-1587UCUUCCCCCUUUUUGGCAUNN 2141 AUGCCAAAAAGGGGGAAGANN 32 1570-1588CUUCCCCCUUUUUGGCAUCNN 2142 GAUGCCAAAAAGGGGGAAGNN 32 1571-1589UUCCCCCUUUUUGGCAUCCNN 2143 GGAUGCCAAAAAGGGGGAANN 32  157-175CCGCUCAUGGUGCCAGCCCNN 2144 GGGCUGGCACCAUGAGCGGNN 32 1572-1590UCCCCCUUUUUGGCAUCCUNN 2145 AGGAUGCCAAAAAGGGGGANN 32 1573-1591CCCCCUUUUUGGCAUCCUGNN 2146 CAGGAUGCCAAAAAGGGGGNN 32 1574-1592CCCCUUUUUGGCAUCCUGGNN 2147 CCAGGAUGCCAAAAAGGGGNN 32 1575-1593CCCUUUUUGGCAUCCUGGCNN 2148 GCCAGGAUGCCAAAAAGGGNN 32 1576-1594CCUUUUUGGCAUCCUGGCUNN 2149 AGCCAGGAUGCCAAAAAGGNN 32 1577-1595CUUUUUGGCAUCCUGGCUUNN 2150 AAGCCAGGAUGCCAAAAAGNN 32 1578-1596UUUUUGGCAUCCUGGCUUGNN 2151 CAAGCCAGGAUGCCAAAAANN 32 1579-1597UUUUGGCAUCCUGGCUUGCNN 2152 GCAAGCCAGGAUGCCAAAANN 32 1580-1598UUUGGCAUCCUGGCUUGCCNN 2153 GGCAAGCCAGGAUGCCAAANN 32 1581-1599UUGGCAUCCUGGCUUGCCUNN 2154 AGGCAAGCCAGGAUGCCAANN 32  158-176CGCUCAUGGUGCCAGCCCANN 2155 UGGGCUGGCACCAUGAGCGNN 32 1582-1600UGGCAUCCUGGCUUGCCUCNN 2156 GAGGCAAGCCAGGAUGCCANN 32 1583-1601GGCAUCCUGGCUUGCCUCCNN 2157 GGAGGCAAGCCAGGAUGCCNN 32 1584-1602GCAUCCUGGCUUGCCUCCANN 2158 UGGAGGCAAGCCAGGAUGCNN 32 1585-1603CAUCCUGGCUUGCCUCCAGNN 2159 CUGGAGGCAAGCCAGGAUGNN 32 1586-1604AUCCUGGCUUGCCUCCAGUNN 2160 ACUGGAGGCAAGCCAGGAUNN 32 1587-1605UCCUGGCUUGCCUCCAGUUNN 2161 AACUGGAGGCAAGCCAGGANN 32 1588-1606CCUGGCUUGCCUCCAGUUUNN 2162 AAACUGGAGGCAAGCCAGGNN 32 1589-1607CUGGCUUGCCUCCAGUUUUNN 2163 AAAACUGGAGGCAAGCCAGNN 32 1590-1608UGGCUUGCCUCCAGUUUUANN 2164 UAAAACUGGAGGCAAGCCANN 32 1591-1609GGCUUGCCUCCAGUUUUAGNN 2165 CUAAAACUGGAGGCAAGCCNN 32  159-177GCUCAUGGUGCCAGCCCAGNN 2166 CUGGGCUGGCACCAUGAGCNN 32 1592-1610GCUUGCCUCCAGUUUUAGGNN 2167 CCUAAAACUGGAGGCAAGCNN 32 1593-1611CUUGCCUCCAGUUUUAGGUNN 2168 ACCUAAAACUGGAGGCAAGNN 32 1594-1612UUGCCUCCAGUUUUAGGUCNN 2169 GACCUAAAACUGGAGGCAANN 32 1595-1613UGCCUCCAGUUUUAGGUCCNN 2170 GGACCUAAAACUGGAGGCANN 32  160-178CUCAUGGUGCCAGCCCAGANN 2171 UCUGGGCUGGCACCAUGAGNN 32  161-179UCAUGGUGCCAGCCCAGAGNN 2172 CUCUGGGCUGGCACCAUGANN 32 1615-1633UUAGUUUGCUUCUGUAAGCNN 2173 GCUUACAGAAGCAAACUAANN 32 1616-1634UAGUUUGCUUCUGUAAGCANN 2174 UGCUUACAGAAGCAAACUANN 32 1617-1635AGUUUGCUUCUGUAAGCAANN 2175 UUGCUUACAGAAGCAAACUNN 32  162-180CAUGGUGCCAGCCCAGAGANN 2176 UCUCUGGGCUGGCACCAUGNN 32  163-181AUGGUGCCAGCCCAGAGAGNN 2177 CUCUCUGGGCUGGCACCAUNN 32 1639-1657GAACACCUGCUGAGGGGGCNN 2178 GCCCCCUCAGCAGGUGUUCNN 32 1640-1658AACACCUGCUGAGGGGGCUNN 2179 AGCCCCCUCAGCAGGUGUUNN 32 1641-1659ACACCUGCUGAGGGGGCUCNN 2180 GAGCCCCCUCAGCAGGUGUNN 32  164-182UGGUGCCAGCCCAGAGAGGNN 2181 CCUCUCUGGGCUGGCACCANN 32 1642-1660CACCUGCUGAGGGGGCUCUNN 2182 AGAGCCCCCUCAGCAGGUGNN 32 1643-1661ACCUGCUGAGGGGGCUCUUNN 2183 AAGAGCCCCCUCAGCAGGUNN 32 1644-1662CCUGCUGAGGGGGCUCUUUNN 2184 AAAGAGCCCCCUCAGCAGGNN 32 1645-1663CUGCUGAGGGGGCUCUUUCNN 2185 GAAAGAGCCCCCUCAGCAGNN 32 1646-1664UGCUGAGGGGGCUCUUUCCNN 2186 GGAAAGAGCCCCCUCAGCANN 32 1647-1665GCUGAGGGGGCUCUUUCCCNN 2187 GGGAAAGAGCCCCCUCAGCNN 32 1648-1666CUGAGGGGGCUCUUUCCCUNN 2188 AGGGAAAGAGCCCCCUCAGNN 32 1649-1667UGAGGGGGCUCUUUCCCUCNN 2189 GAGGGAAAGAGCCCCCUCANN 32 1650-1668GAGGGGGCUCUUUCCCUCANN 2190 UGAGGGAAAGAGCCCCCUCNN 32  165-183GGUGCCAGCCCAGAGAGGGNN 2191 CCCUCUCUGGGCUGGCACCNN 32  166-184GUGCCAGCCCAGAGAGGGGNN 2192 CCCCUCUCUGGGCUGGCACNN 32 1670-1688GUAUACUUCAAGUAAGAUCNN 2193 GAUCUUACUUGAAGUAUACNN 32 1671-1689UAUACUUCAAGUAAGAUCANN 2194 UGAUCUUACUUGAAGUAUANN 32  167-185UGCCAGCCCAGAGAGGGGCNN 2195 GCCCCUCUCUGGGCUGGCANN 32 1672-1690AUACUUCAAGUAAGAUCAANN 2196 UUGAUCUUACUUGAAGUAUNN 32 1673-1691UACUUCAAGUAAGAUCAAGNN 2197 CUUGAUCUUACUUGAAGUANN 32 1674-1692ACUUCAAGUAAGAUCAAGANN 2198 UCUUGAUCUUACUUGAAGUNN 32 1675-1693CUUCAAGUAAGAUCAAGAANN 2199 UUCUUGAUCUUACUUGAAGNN 32 1676-1694UUCAAGUAAGAUCAAGAAUNN 2200 AUUCUUGAUCUUACUUGAANN 32 1677-1695UCAAGUAAGAUCAAGAAUCNN 2201 GAUUCUUGAUCUUACUUGANN 32 1678-1696CAAGUAAGAUCAAGAAUCUNN 2202 AGAUUCUUGAUCUUACUUGNN 32 1679-1697AAGUAAGAUCAAGAAUCUUNN 2203 AAGAUUCUUGAUCUUACUUNN 32 1680-1698AGUAAGAUCAAGAAUCUUUNN 2204 AAAGAUUCUUGAUCUUACUNN 32 1681-1699GUAAGAUCAAGAAUCUUUUNN 2205 AAAAGAUUCUUGAUCUUACNN 32 1682-1700UAAGAUCAAGAAUCUUUUGNN 2206 CAAAAGAUUCUUGAUCUUANN 32 1683-1701AAGAUCAAGAAUCUUUUGUNN 2207 ACAAAAGAUUCUUGAUCUUNN 32 1684-1702AGAUCAAGAAUCUUUUGUGNN 2208 CACAAAAGAUUCUUGAUCUNN 32 1685-1703GAUCAAGAAUCUUUUGUGANN 2209 UCACAAAAGAUUCUUGAUCNN 32 1686-1704AUCAAGAAUCUUUUGUGAANN 2210 UUCACAAAAGAUUCUUGAUNN 32 1687-1705UCAAGAAUCUUUUGUGAAANN 2211 UUUCACAAAAGAUUCUUGANN 32 1707-1725UAUAGAAAUUUACUAUGUANN 2212 UACAUAGUAAAUUUCUAUANN 32 1708-1726AUAGAAAUUUACUAUGUAANN 2213 UUACAUAGUAAAUUUCUAUNN 32 1709-1727UAGAAAUUUACUAUGUAAANN 2214 UUUACAUAGUAAAUUUCUANN 32 1710-1728AGAAAUUUACUAUGUAAAUNN 2215 AUUUACAUAGUAAAUUUCUNN 32 1711-1729GAAAUUUACUAUGUAAAUGNN 2216 CAUUUACAUAGUAAAUUUCNN 32 1712-1730AAAUUUACUAUGUAAAUGCNN 2217 GCAUUUACAUAGUAAAUUUNN 32 1713-1731AAUUUACUAUGUAAAUGCUNN 2218 AGCAUUUACAUAGUAAAUUNN 32 1714-1732AUUUACUAUGUAAAUGCUUNN 2219 AAGCAUUUACAUAGUAAAUNN 32 1715-1733UUUACUAUGUAAAUGCUUGNN 2220 CAAGCAUUUACAUAGUAAANN 32 1716-1734UUACUAUGUAAAUGCUUGANN 2221 UCAAGCAUUUACAUAGUAANN 32 1717-1735UACUAUGUAAAUGCUUGAUNN 2222 AUCAAGCAUUUACAUAGUANN 32 1718-1736ACUAUGUAAAUGCUUGAUGNN 2223 CAUCAAGCAUUUACAUAGUNN 32 1719-1737CUAUGUAAAUGCUUGAUGGNN 2224 CCAUCAAGCAUUUACAUAGNN 32 1720-1738UAUGUAAAUGCUUGAUGGANN 2225 UCCAUCAAGCAUUUACAUANN 32 1721-1739AUGUAAAUGCUUGAUGGAANN 2226 UUCCAUCAAGCAUUUACAUNN 32 1722-1740UGUAAAUGCUUGAUGGAAUNN 2227 AUUCCAUCAAGCAUUUACANN 32 1723-1741GUAAAUGCUUGAUGGAAUUNN 2228 AAUUCCAUCAAGCAUUUACNN 32 1724-1742UAAAUGCUUGAUGGAAUUUNN 2229 AAAUUCCAUCAAGCAUUUANN 32 1725-1743AAAUGCUUGAUGGAAUUUUNN 2230 AAAAUUCCAUCAAGCAUUUNN 32 1726-1744AAUGCUUGAUGGAAUUUUUNN 2231 AAAAAUUCCAUCAAGCAUUNN 32 1727-1745AUGCUUGAUGGAAUUUUUUNN 2232 AAAAAAUUCCAUCAAGCAUNN 32 1728-1746UGCUUGAUGGAAUUUUUUCNN 2233 GAAAAAAUUCCAUCAAGCANN 32 1729-1747GCUUGAUGGAAUUUUUUCCNN 2234 GGAAAAAAUUCCAUCAAGCNN 32 1730-1748CUUGAUGGAAUUUUUUCCUNN 2235 AGGAAAAAAUUCCAUCAAGNN 32 1731-1749UUGAUGGAAUUUUUUCCUGNN 2236 CAGGAAAAAAUUCCAUCAANN 32 1732-1750UGAUGGAAUUUUUUCCUGCNN 2237 GCAGGAAAAAAUUCCAUCANN 32 1733-1751GAUGGAAUUUUUUCCUGCUNN 2238 AGCAGGAAAAAAUUCCAUCNN 32 1734-1752AUGGAAUUUUUUCCUGCUANN 2239 UAGCAGGAAAAAAUUCCAUNN 32 1735-1753UGGAAUUUUUUCCUGCUAGNN 2240 CUAGCAGGAAAAAAUUCCANN 32 1736-1754GGAAUUUUUUCCUGCUAGUNN 2241 ACUAGCAGGAAAAAAUUCCNN 33 1737-1755GAAUUUUUUCCUGCUAGUGNN 2242 CACUAGCAGGAAAAAAUUCNN 33 1738-1756AAUUUUUUCCUGCUAGUGUNN 2243 ACACUAGCAGGAAAAAAUUNN 33 1739-1757AUUUUUUCCUGCUAGUGUANN 2244 UACACUAGCAGGAAAAAAUNN 33 1740-1758UUUUUUCCUGCUAGUGUAGNN 2245 CUACACUAGCAGGAAAAAANN 33 1741-1759UUUUUCCUGCUAGUGUAGCNN 2246 GCUACACUAGCAGGAAAAANN 33 1742-1760UUUUCCUGCUAGUGUAGCUNN 2247 AGCUACACUAGCAGGAAAANN 33 1743-1761UUUCCUGCUAGUGUAGCUUNN 2248 AAGCUACACUAGCAGGAAANN 33 1744-1762UUCCUGCUAGUGUAGCUUCNN 2249 GAAGCUACACUAGCAGGAANN 33 1745-1763UCCUGCUAGUGUAGCUUCUNN 2250 AGAAGCUACACUAGCAGGANN 33 1746-1764CCUGCUAGUGUAGCUUCUGNN 2251 CAGAAGCUACACUAGCAGGNN 33 1747-1765CUGCUAGUGUAGCUUCUGANN 2252 UCAGAAGCUACACUAGCAGNN 33 1748-1766UGCUAGUGUAGCUUCUGAANN 2253 UUCAGAAGCUACACUAGCANN 33 1749-1767GCUAGUGUAGCUUCUGAAANN 2254 UUUCAGAAGCUACACUAGCNN 33 1750-1768CUAGUGUAGCUUCUGAAAGNN 2255 CUUUCAGAAGCUACACUAGNN 33 1751-1769UAGUGUAGCUUCUGAAAGGNN 2256 CCUUUCAGAAGCUACACUANN 33 1752-1770AGUGUAGCUUCUGAAAGGUNN 2257 ACCUUUCAGAAGCUACACUNN 33 1753-1771GUGUAGCUUCUGAAAGGUGNN 2258 CACCUUUCAGAAGCUACACNN 33 1754-1772UGUAGCUUCUGAAAGGUGCNN 2259 GCACCUUUCAGAAGCUACANN 33 1755-1773GUAGCUUCUGAAAGGUGCUNN 2260 AGCACCUUUCAGAAGCUACNN 33 1756-1774UAGCUUCUGAAAGGUGCUUNN 2261 AAGCACCUUUCAGAAGCUANN 33 1757-1775AGCUUCUGAAAGGUGCUUUNN 2262 AAAGCACCUUUCAGAAGCUNN 33 1758-1776GCUUCUGAAAGGUGCUUUCNN 2263 GAAAGCACCUUUCAGAAGCNN 33 1777-1795UCCAUUUAUUUAAAACUACNN 2264 GUAGUUUUAAAUAAAUGGANN 33 1778-1796CCAUUUAUUUAAAACUACCNN 2265 GGUAGUUUUAAAUAAAUGGNN 33 1779-1797CAUUUAUUUAAAACUACCCNN 2266 GGGUAGUUUUAAAUAAAUGNN 33 1780-1798AUUUAUUUAAAACUACCCANN 2267 UGGGUAGUUUUAAAUAAAUNN 33 1781-1799UUUAUUUAAAACUACCCAUNN 2268 AUGGGUAGUUUUAAAUAAANN 33 1782-1800UUAUUUAAAACUACCCAUGNN 2269 CAUGGGUAGUUUUAAAUAANN 33 1783-1801UAUUUAAAACUACCCAUGCNN 2270 GCAUGGGUAGUUUUAAAUANN 33 1784-1802AUUUAAAACUACCCAUGCANN 2271 UGCAUGGGUAGUUUUAAAUNN 33 1785-1803UUUAAAACUACCCAUGCAANN 2272 UUGCAUGGGUAGUUUUAAANN 33 1786-1804UUAAAACUACCCAUGCAAUNN 2273 AUUGCAUGGGUAGUUUUAANN 33 1787-1805UAAAACUACCCAUGCAAUUNN 2274 AAUUGCAUGGGUAGUUUUANN 33 1788-1806AAAACUACCCAUGCAAUUANN 2275 UAAUUGCAUGGGUAGUUUUNN 33 1789-1807AAACUACCCAUGCAAUUAANN 2276 UUAAUUGCAUGGGUAGUUUNN 33 1790-1808AACUACCCAUGCAAUUAAANN 2277 UUUAAUUGCAUGGGUAGUUNN 33 1791-1809ACUACCCAUGCAAUUAAAANN 2278 UUUUAAUUGCAUGGGUAGUNN 33 1792-1810CUACCCAUGCAAUUAAAAGNN 2279 CUUUUAAUUGCAUGGGUAGNN 33 1793-1811UACCCAUGCAAUUAAAAGGNN 2280 CCUUUUAAUUGCAUGGGUANN 33 1794-1812ACCCAUGCAAUUAAAAGGUNN 2281 ACCUUUUAAUUGCAUGGGUNN 33 1795-1813CCCAUGCAAUUAAAAGGUANN 2282 UACCUUUUAAUUGCAUGGGNN 33 1796-1814CCAUGCAAUUAAAAGGUACNN 2283 GUACCUUUUAAUUGCAUGGNN 33 1797-1815CAUGCAAUUAAAAGGUACANN 2284 UGUACCUUUUAAUUGCAUGNN 33 1798-1816AUGCAAUUAAAAGGUACAANN 2285 UUGUACCUUUUAAUUGCAUNN 33 1799-1817UGCAAUUAAAAGGUACAAUNN 2286 AUUGUACCUUUUAAUUGCANN 33 1800-1818GCAAUUAAAAGGUACAAUGNN 2287 CAUUGUACCUUUUAAUUGCNN 33 1801-1819CAAUUAAAAGGUACAAUGCNN 2288 GCAUUGUACCUUUUAAUUGNN 33 1802-1820AAUUAAAAGGUACAAUGCANN 2289 UGCAUUGUACCUUUUAAUUNN 33  187-205AGCCCGGAGGCAGCGAGCGNN 2290 CGCTCGCTGCCTCCGGGCTNN 33  188-206GCCCGGAGGCAGCGAGCGGNN 2291 CCGCTCGCTGCCTCCGGGCNN 33  189-207CCCGGAGGCAGCGAGCGGGNN 2292 CCCGCTCGCTGCCTCCGGGNN 33  190-208CCGGAGGCAGCGAGCGGGGNN 2293 CCCCGCTCGCTGCCTCCGGNN 33  191-209CGGAGGCAGCGAGCGGGGGNN 2294 CCCCCGCTCGCTGCCTCCGNN 33  192-210GGAGGCAGCGAGCGGGGGGNN 2295 CCCCCCGCTCGCTGCCTCCNN 33  193-211GAGGCAGCGAGCGGGGGGCNN 2296 GCCCCCCGCTCGCTGCCTCNN 33  194-212AGGCAGCGAGCGGGGGGCUNN 2297 AGCCCCCCGCUCGCUGCCUNN 33  195-213GGCAGCGAGCGGGGGGCUGNN 2298 CAGCCCCCCGCUCGCUGCCNN 33  196-214GCAGCGAGCGGGGGGCUGCNN 2299 GCAGCCCCCCGCUCGCUGCNN 33  197-215CAGCGAGCGGGGGGCUGCCNN 2300 GGCAGCCCCCCGCUCGCUGNN 33  198-216AGCGAGCGGGGGGCUGCCCNN 2301 GGGCAGCCCCCCGCUCGCUNN 33  199-217GCGAGCGGGGGGCUGCCCCNN 2302 GGGGCAGCCCCCCGCUCGCNN 33  200-218CGAGCGGGGGGCUGCCCCANN 2303 UGGGGCAGCCCCCCGCUCGNN 33  201-219GAGCGGGGGGCUGCCCCAGNN 2304 CUGGGGCAGCCCCCCGCUCNN 33  202-220AGCGGGGGGCUGCCCCAGGNN 2305 CCUGGGGCAGCCCCCCGCUNN 33  203-221GCGGGGGGCUGCCCCAGGCNN 2306 GCCUGGGGCAGCCCCCCGCNN 33  204-222CGGGGGGCUGCCCCAGGCGNN 2307 CGCCUGGGGCAGCCCCCCGNN 33  205-223GGGGGGCUGCCCCAGGCGCNN 2308 GCGCCUGGGGCAGCCCCCCNN 33  206-224GGGGGCUGCCCCAGGCGCGNN 2309 CGCGCCUGGGGCAGCCCCCNN 33  207-225GGGGCUGCCCCAGGCGCGCNN 2310 GCGCGCCUGGGGCAGCCCCNN 33  208-226GGGCUGCCCCAGGCGCGCANN 2311 UGCGCGCCUGGGGCAGCCCNN 33  209-227GGCUGCCCCAGGCGCGCAANN 2312 UUGCGCGCCUGGGGCAGCCNN 33  210-228GCUGCCCCAGGCGCGCAAGNN 2313 CUUGCGCGCCUGGGGCAGCNN 33  211-229CUGCCCCAGGCGCGCAAGCNN 2314 GCUUGCGCGCCUGGGGCAGNN 33  212-230UGCCCCAGGCGCGCAAGCGNN 2315 CGCUUGCGCGCCUGGGGCANN 33  247-265CUGAGCCCCGAGGAGAAGGNN 2316 CCUUCUCCUCGGGGCUCAGNN 33  248-266UGAGCCCCGAGGAGAAGGCNN 2317 GCCUUCUCCUCGGGGCUCANN 33  249-267GAGCCCCGAGGAGAAGGCGNN 2318 CGCCTTCTCCTCGGGGCTCNN 33  250-268AGCCCCGAGGAGAAGGCGCNN 2319 GCGCCTTCTCCTCGGGGCTNN 33  251-269GCCCCGAGGAGAAGGCGCUNN 2320 AGCGCCUUCUCCUCGGGGCNN 33  252-270CCCCGAGGAGAAGGCGCUGNN 2321 CAGCGCCUUCUCCUCGGGGNN 33  253-271CCCGAGGAGAAGGCGCUGANN 2322 UCAGCGCCUUCUCCUCGGGNN 33  254-272CCGAGGAGAAGGCGCUGAGNN 2323 CUCAGCGCCUUCUCCUCGGNN 33  255-273CGAGGAGAAGGCGCUGAGGNN 2324 CCUCAGCGCCUUCUCCUCGNN 33  256-274GAGGAGAAGGCGCUGAGGANN 2325 UCCUCAGCGCCUUCUCCUCNN 33  257-275AGGAGAAGGCGCUGAGGAGNN 2326 CUCCUCAGCGCCUUCUCCUNN 33  258-276GGAGAAGGCGCUGAGGAGGNN 2327 CCUCCUCAGCGCCUUCUCCNN 33  259-277GAGAAGGCGCUGAGGAGGANN 2328 UCCUCCUCAGCGCCUUCUCNN 33  260-278AGAAGGCGCUGAGGAGGAANN 2329 UUCCUCCUCAGCGCCUUCUNN 33  261-279GAAGGCGCUGAGGAGGAAANN 2330 UUUCCUCCUCAGCGCCUUCNN 33  262-280AAGGCGCUGAGGAGGAAACNN 2331 GUUUCCUCCUCAGCGCCUUNN 33  263-281AGGCGCUGAGGAGGAAACUNN 2332 AGUUUCCUCCUCAGCGCCUNN 33  264-282GGCGCUGAGGAGGAAACUGNN 2333 CAGUUUCCUCCUCAGCGCCNN 33  265-283GCGCUGAGGAGGAAACUGANN 2334 UCAGUUUCCUCCUCAGCGCNN 33  266-284CGCUGAGGAGGAAACUGAANN 2335 UUCAGUUUCCUCCUCAGCGNN 33  267-285GCUGAGGAGGAAACUGAAANN 2336 UUUCAGUUUCCUCCUCAGCNN 33  268-286CUGAGGAGGAAACUGAAAANN 2337 UUUUCAGUUUCCUCCUCAGNN 33  269-287UGAGGAGGAAACUGAAAAANN 2338 UUUUUCAGUUUCCUCCUCANN 33  270-288GAGGAGGAAACUGAAAAACNN 2339 GUUUUUCAGUUUCCUCCUCNN 33  271-289AGGAGGAAACUGAAAAACANN 2340 UGUUUUUCAGUUUCCUCCUNN 33  272-290GGAGGAAACUGAAAAACAGNN 2341 CUGUUUUUCAGUUUCCUCCNN 34  273-291GAGGAAACUGAAAAACAGANN 2342 UCUGUUUUUCAGUUUCCUCNN 34  274-292AGGAAACUGAAAAACAGAGNN 2343 CUCUGUUUUUCAGUUUCCUNN 34  275-293GGAAACUGAAAAACAGAGUNN 2344 ACUCUGUUUUUCAGUUUCCNN 34  276-294GAAACUGAAAAACAGAGUANN 2345 UACUCUGUUUUUCAGUUUCNN 34  277-295AAACUGAAAAACAGAGUAGNN 2346 CUACUCUGUUUUUCAGUUUNN 34  278-296AACUGAAAAACAGAGUAGCNN 2347 GCUACUCUGUUUUUCAGUUNN 34  279-297ACUGAAAAACAGAGUAGCANN 2348 UGCUACUCUGUUUUUCAGUNN 34  280-298CUGAAAAACAGAGUAGCAGNN 2349 CUGCUACUCUGUUUUUCAGNN 34  281-299UGAAAAACAGAGUAGCAGCNN 2350 GCUGCUACUCUGUUUUUCANN 34  282-300GAAAAACAGAGUAGCAGCUNN 2351 AGCUGCUACUCUGUUUUUCNN 34  283-301AAAAACAGAGUAGCAGCUCNN 2352 GAGCUGCUACUCUGUUUUUNN 34  284-302AAAACAGAGUAGCAGCUCANN 2353 UGAGCUGCUACUCUGUUUUNN 34  285-303AAACAGAGUAGCAGCUCAGNN 2354 CUGAGCUGCUACUCUGUUUNN 34  286-304AACAGAGUAGCAGCUCAGANN 2355 UCUGAGCUGCUACUCUGUUNN 34  287-305ACAGAGUAGCAGCUCAGACNN 2356 GUCUGAGCUGCUACUCUGUNN 34  288-306CAGAGUAGCAGCUCAGACUNN 2357 AGUCUGAGCUGCUACUCUGNN 34  289-307AGAGUAGCAGCUCAGACUGNN 2358 CAGUCUGAGCUGCUACUCUNN 34  290-308GAGUAGCAGCUCAGACUGCNN 2359 GCAGUCUGAGCUGCUACUCNN 34  291-309AGUAGCAGCUCAGACUGCCNN 2360 GGCAGUCUGAGCUGCUACUNN 34  292-310GUAGCAGCUCAGACUGCCANN 2361 UGGCAGUCUGAGCUGCUACNN 34  293-311UAGCAGCUCAGACUGCCAGNN 2362 CUGGCAGUCUGAGCUGCUANN 34  294-312AGCAGCUCAGACUGCCAGANN 2363 UCUGGCAGUCUGAGCUGCUNN 34  295-313GCAGCUCAGACUGCCAGAGNN 2364 CUCUGGCAGUCUGAGCUGCNN 34  296-314CAGCUCAGACUGCCAGAGANN 2365 UCUCUGGCAGUCUGAGCUGNN 34  297-315AGCUCAGACUGCCAGAGAUNN 2366 AUCUCUGGCAGUCUGAGCUNN 34  298-316GCUCAGACUGCCAGAGAUCNN 2367 GAUCUCUGGCAGUCUGAGCNN 34  299-317CUCAGACUGCCAGAGAUCGNN 2368 CGAUCUCUGGCAGUCUGAGNN 34  300-318UCAGACUGCCAGAGAUCGANN 2369 UCGAUCUCUGGCAGUCUGANN 34  301-319CAGACUGCCAGAGAUCGAANN 2370 UUCGAUCUCUGGCAGUCUGNN 34  302-320AGACUGCCAGAGAUCGAAANN 2371 UUUCGAUCUCUGGCAGUCUNN 34  303-321GACUGCCAGAGAUCGAAAGNN 2372 CUUUCGAUCUCUGGCAGUCNN 34  304-322ACUGCCAGAGAUCGAAAGANN 2373 UCUUUCGAUCUCUGGCAGUNN 34  305-323CUGCCAGAGAUCGAAAGAANN 2374 UUCUUUCGAUCUCUGGCAGNN 34  325-343GCUCGAAUGAGUGAGCUGGNN 2375 CCAGCUCACUCAUUCGAGCNN 34  326-344CUCGAAUGAGUGAGCUGGANN 2376 UCCAGCUCACUCAUUCGAGNN 34  327-345UCGAAUGAGUGAGCUGGAANN 2377 UUCCAGCUCACUCAUUCGANN 34  328-346CGAAUGAGUGAGCUGGAACNN 2378 GUUCCAGCUCACUCAUUCGNN 34  329-347GAAUGAGUGAGCUGGAACANN 2379 UGUUCCAGCUCACUCAUUCNN 34  330-348AAUGAGUGAGCUGGAACAGNN 2380 CUGUUCCAGCUCACUCAUUNN 34  331-349AUGAGUGAGCUGGAACAGCNN 2381 GCUGUUCCAGCUCACUCAUNN 34  332-350UGAGUGAGCUGGAACAGCANN 2382 UGCUGUUCCAGCUCACUCANN 34  333-351GAGUGAGCUGGAACAGCAANN 2383 UUGCUGUUCCAGCUCACUCNN 34  334-352AGUGAGCUGGAACAGCAAGNN 2384 CUUGCUGUUCCAGCUCACUNN 34  335-353GUGAGCUGGAACAGCAAGUNN 2385 ACUUGCUGUUCCAGCUCACNN 34  336-354UGAGCUGGAACAGCAAGUGNN 2386 CACUUGCUGUUCCAGCUCANN 34  337-355GAGCUGGAACAGCAAGUGGNN 2387 CCACUUGCUGUUCCAGCUCNN 34  338-356AGCUGGAACAGCAAGUGGUNN 2388 ACCACUUGCUGUUCCAGCUNN 34  339-357GCUGGAACAGCAAGUGGUANN 2389 UACCACUUGCUGUUCCAGCNN 34  340-358CUGGAACAGCAAGUGGUAGNN 2390 CUACCACUUGCUGUUCCAGNN 34  341-359UGGAACAGCAAGUGGUAGANN 2391 UCUACCACUUGCUGUUCCANN 34  342-360GGAACAGCAAGUGGUAGAUNN 2392 AUCUACCACUUGCUGUUCCNN 34  343-361GAACAGCAAGUGGUAGAUUNN 2393 AAUCUACCACUUGCUGUUCNN 34  344-362AACAGCAAGUGGUAGAUUUNN 2394 AAAUCUACCACUUGCUGUUNN 34  345-363ACAGCAAGUGGUAGAUUUANN 2395 UAAAUCUACCACUUGCUGUNN 34  346-364CAGCAAGUGGUAGAUUUAGNN 2396 CUAAAUCUACCACUUGCUGNN 34  347-365AGCAAGUGGUAGAUUUAGANN 2397 UCUAAAUCUACCACUUGCUNN 34  348-366GCAAGUGGUAGAUUUAGAANN 2398 UUCUAAAUCUACCACUUGCNN 34  349-367CAAGUGGUAGAUUUAGAAGNN 2399 CUUCUAAAUCUACCACUUGNN 34  350-368AAGUGGUAGAUUUAGAAGANN 2400 UCUUCUAAAUCUACCACUUNN 34  351-369AGUGGUAGAUUUAGAAGAANN 2401 UUCUUCUAAAUCUACCACUNN 34  352-370GUGGUAGAUUUAGAAGAAGNN 2402 CUUCUUCUAAAUCUACCACNN 34  353-371UGGUAGAUUUAGAAGAAGANN 2403 UCUUCUUCUAAAUCUACCANN 34  354-372GGUAGAUUUAGAAGAAGAGNN 2404 CUCUUCUUCUAAAUCUACCNN 34  355-373GUAGAUUUAGAAGAAGAGANN 2405 UCUCUUCUUCUAAAUCUACNN 34  356-374UAGAUUUAGAAGAAGAGAANN 2406 UUCUCUUCUUCUAAAUCUANN 34  357-375AGAUUUAGAAGAAGAGAACNN 2407 GUUCUCUUCUUCUAAAUCUNN 34  358-376GAUUUAGAAGAAGAGAACCNN 2408 GGUUCUCUUCUUCUAAAUCNN 34  359-377AUUUAGAAGAAGAGAACCANN 2409 UGGUUCUCUUCUUCUAAAUNN 34  360-378UUUAGAAGAAGAGAACCAANN 2410 UUGGUUCUCUUCUUCUAAANN 34  361-379UUAGAAGAAGAGAACCAAANN 2411 UUUGGUUCUCUUCUUCUAANN 34  362-380UAGAAGAAGAGAACCAAAANN 2412 UUUUGGUUCUCUUCUUCUANN 34  363-381AGAAGAAGAGAACCAAAAANN 2413 TTTTTGGTTCTCTTCTTCTNN 34  364-382GAAGAAGAGAACCAAAAACNN 2414 GTTTTTGGTTCTCTTCTTCNN 34  365-383AAGAAGAGAACCAAAAACUNN 2415 AGUUUUUGGUUCUCUUCUUNN 34  366-384AGAAGAGAACCAAAAACUUNN 2416 AAGUUUUUGGUUCUCUUCUNN 34  367-385GAAGAGAACCAAAAACUUUNN 2417 AAAGUUUUUGGUUCUCUUCNN 34  368-386AAGAGAACCAAAAACUUUUNN 2418 AAAAGUUUUUGGUUCUCUUNN 34  369-387AGAGAACCAAAAACUUUUGNN 2419 CAAAAGUUUUUGGUUCUCUNN 34  370-388GAGAACCAAAAACUUUUGCNN 2420 GCAAAAGUUUUUGGUUCUCNN 34  371-389AGAACCAAAAACUUUUGCUNN 2421 AGCAAAAGUUUUUGGUUCUNN 34  372-390GAACCAAAAACUUUUGCUANN 2422 UAGCAAAAGUUUUUGGUUCNN 34  373-391AACCAAAAACUUUUGCUAGNN 2423 CUAGCAAAAGUUUUUGGUUNN 34  374-392ACCAAAAACUUUUGCUAGANN 2424 UCUAGCAAAAGUUUUUGGUNN 34  375-393CCAAAAACUUUUGCUAGAANN 2425 UUCUAGCAAAAGUUUUUGGNN 34  376-394CAAAAACUUUUGCUAGAAANN 2426 UUUCUAGCAAAAGUUUUUGNN 34  377-395AAAAACUUUUGCUAGAAAANN 2427 UUUUCUAGCAAAAGUUUUUNN 34  378-396AAAACUUUUGCUAGAAAAUNN 2428 AUUUUCUAGCAAAAGUUUUNN 34  379-397AAACUUUUGCUAGAAAAUCNN 2429 GAUUUUCUAGCAAAAGUUUNN 34  380-398AACUUUUGCUAGAAAAUCANN 2430 UGAUUUUCUAGCAAAAGUUNN 34  381-399ACUUUUGCUAGAAAAUCAGNN 2431 CUGAUUUUCUAGCAAAAGUNN 34  382-400CUUUUGCUAGAAAAUCAGCNN 2432 GCUGAUUUUCUAGCAAAAGNN 34  383-401UUUUGCUAGAAAAUCAGCUNN 2433 AGCUGAUUUUCUAGCAAAANN 34  384-402UUUGCUAGAAAAUCAGCUUNN 2434 AAGCUGAUUUUCUAGCAAANN 34  385-403UUGCUAGAAAAUCAGCUUUNN 2435 AAAGCUGAUUUUCUAGCAANN 34  386-404UGCUAGAAAAUCAGCUUUUNN 2436 AAAAGCUGAUUUUCUAGCANN 34  387-405GCUAGAAAAUCAGCUUUUANN 2437 UAAAAGCUGAUUUUCUAGCNN 34  388-406CUAGAAAAUCAGCUUUUACNN 2438 GUAAAAGCUGAUUUUCUAGNN 34  389-407UAGAAAAUCAGCUUUUACGNN 2439 CGUAAAAGCUGAUUUUCUANN 34  390-408AGAAAAUCAGCUUUUACGANN 2440 UCGUAAAAGCUGAUUUUCUNN 34  391-409GAAAAUCAGCUUUUACGAGNN 2441 CUCGUAAAAGCUGAUUUUCNN 35  392-410AAAAUCAGCUUUUACGAGANN 2442 UCUCGUAAAAGCUGAUUUUNN 35  393-411AAAUCAGCUUUUACGAGAGNN 2443 CUCUCGUAAAAGCUGAUUUNN 35  394-412AAUCAGCUUUUACGAGAGANN 2444 UCUCUCGUAAAAGCUGAUUNN 35  395-413AUCAGCUUUUACGAGAGAANN 2445 UUCUCUCGUAAAAGCUGAUNN 35  396-414UCAGCUUUUACGAGAGAAANN 2446 UUUCUCUCGUAAAAGCUGANN 35  397-415CAGCUUUUACGAGAGAAAANN 2447 UUUUCUCUCGUAAAAGCUGNN 35  398-416AGCUUUUACGAGAGAAAACNN 2448 GUUUUCUCUCGUAAAAGCUNN 35  399-417GCUUUUACGAGAGAAAACUNN 2449 AGUUUUCUCUCGUAAAAGCNN 35  400-418CUUUUACGAGAGAAAACUCNN 2450 GAGUUUUCUCUCGUAAAAGNN 35  401-419UUUUACGAGAGAAAACUCANN 2451 UGAGUUUUCUCUCGUAAAANN 35  421-439GGCCUUGUAGUUGAGAACCNN 2452 GGUUCUCAACUACAAGGCCNN 35  422-440GCCUUGUAGUUGAGAACCANN 2453 UGGUUCUCAACUACAAGGCNN 35  423-441CCUUGUAGUUGAGAACCAGNN 2454 CUGGUUCUCAACUACAAGGNN 35  424-442CUUGUAGUUGAGAACCAGGNN 2455 CCUGGUUCUCAACUACAAGNN 35  425-443UUGUAGUUGAGAACCAGGANN 2456 UCCUGGUUCUCAACUACAANN 35  426-444UGUAGUUGAGAACCAGGAGNN 2457 CUCCUGGUUCUCAACUACANN 35  427-445GUAGUUGAGAACCAGGAGUNN 2458 ACUCCUGGUUCUCAACUACNN 35  428-446UAGUUGAGAACCAGGAGUUNN 2459 AACUCCUGGUUCUCAACUANN 35  429-447AGUUGAGAACCAGGAGUUANN 2460 UAACUCCUGGUUCUCAACUNN 35  430-448GUUGAGAACCAGGAGUUAANN 2461 UUAACUCCUGGUUCUCAACNN 35  431-449UUGAGAACCAGGAGUUAAGNN 2462 CUUAACUCCUGGUUCUCAANN 35  432-450UGAGAACCAGGAGUUAAGANN 2463 UCUUAACUCCUGGUUCUCANN 35  433-451GAGAACCAGGAGUUAAGACNN 2464 GUCUUAACUCCUGGUUCUCNN 35  434-452AGAACCAGGAGUUAAGACANN 2465 UGUCUUAACUCCUGGUUCUNN 35  435-453GAACCAGGAGUUAAGACAGNN 2466 CUGUCUUAACUCCUGGUUCNN 35  436-454AACCAGGAGUUAAGACAGCNN 2467 GCUGUCUUAACUCCUGGUUNN 35  437-455ACCAGGAGUUAAGACAGCGNN 2468 CGCUGUCUUAACUCCUGGUNN 35  438-456CCAGGAGUUAAGACAGCGCNN 2469 GCGCUGUCUUAACUCCUGGNN 35   44-62GAGCUAUGGUGGUGGUGGCNN 2470 GCCACCACCACCAUAGCUCNN 35   45-63AGCUAUGGUGGUGGUGGCANN 2471 UGCCACCACCACCAUAGCUNN 35  458-476UGGGGAUGGAUGCCCUGGUNN 2472 ACCAGGGCAUCCAUCCCCANN 35  459-477GGGGAUGGAUGCCCUGGUUNN 2473 AACCAGGGCAUCCAUCCCCNN 35  460-478GGGAUGGAUGCCCUGGUUGNN 2474 CAACCAGGGCAUCCAUCCCNN 35  461-479GGAUGGAUGCCCUGGUUGCNN 2475 GCAACCAGGGCAUCCAUCCNN 35  462-480GAUGGAUGCCCUGGUUGCUNN 2476 AGCAACCAGGGCAUCCAUCNN 35   46-64GCUAUGGUGGUGGUGGCAGNN 2477 CUGCCACCACCACCAUAGCNN 35   47-65CUAUGGUGGUGGUGGCAGCNN 2478 GCUGCCACCACCACCAUAGNN 35  482-500AAGAGGAGGCGGAAGCCAANN 2479 TTGGCTTCCGCCTCCTCTTNN 35  483-501AGAGGAGGCGGAAGCCAAGNN 2480 CTTGGCTTCCGCCTCCTCTNN 35  484-502GAGGAGGCGGAAGCCAAGGNN 2481 CCTTGGCTTCCGCCTCCTCNN 35  485-503AGGAGGCGGAAGCCAAGGGNN 2482 CCCTTGGCTTCCGCCTCCTNN 35  486-504GGAGGCGGAAGCCAAGGGGNN 2483 CCCCTTGGCTTCCGCCTCCNN 35   48-66UAUGGUGGUGGUGGCAGCCNN 2484 GGCUGCCACCACCACCAUANN 35  487-505GAGGCGGAAGCCAAGGGGANN 2485 TCCCCTTGGCTTCCGCCTCNN 35  488-506AGGCGGAAGCCAAGGGGAANN 2486 TTCCCCTTGGCTTCCGCCTNN 35  489-507GGCGGAAGCCAAGGGGAAUNN 2487 AUUCCCCUUGGCUUCCGCCNN 35  490-508GCGGAAGCCAAGGGGAAUGNN 2488 CAUUCCCCUUGGCUUCCGCNN 35   49-67AUGGUGGUGGUGGCAGCCGNN 2489 CGGCUGCCACCACCACCAUNN 35   50-68UGGUGGUGGUGGCAGCCGCNN 2490 GCGGCUGCCACCACCACCANN 35  510-528AGUGAGGCCAGUGGCCGGGNN 2491 CCCGGCCACUGGCCUCACUNN 35  511-529GUGAGGCCAGUGGCCGGGUNN 2492 ACCCGGCCACUGGCCUCACNN 35  512-530UGAGGCCAGUGGCCGGGUCNN 2493 GACCCGGCCACUGGCCUCANN 35  513-531GAGGCCAGUGGCCGGGUCUNN 2494 AGACCCGGCCACUGGCCUCNN 35  514-532AGGCCAGUGGCCGGGUCUGNN 2495 CAGACCCGGCCACUGGCCUNN 35  515-533GGCCAGUGGCCGGGUCUGCNN 2496 GCAGACCCGGCCACUGGCCNN 35  516-534GCCAGUGGCCGGGUCUGCUNN 2497 AGCAGACCCGGCCACUGGCNN 35  517-535CCAGUGGCCGGGUCUGCUGNN 2498 CAGCAGACCCGGCCACUGGNN 35  518-536CAGUGGCCGGGUCUGCUGANN 2499 UCAGCAGACCCGGCCACUGNN 35  519-537AGUGGCCGGGUCUGCUGAGNN 2500 CUCAGCAGACCCGGCCACUNN 35  520-538GUGGCCGGGUCUGCUGAGUNN 2501 ACUCAGCAGACCCGGCCACNN 35  521-539UGGCCGGGUCUGCUGAGUCNN 2502 GACUCAGCAGACCCGGCCANN 35  522-540GGCCGGGUCUGCUGAGUCCNN 2503 GGACUCAGCAGACCCGGCCNN 35  523-541GCCGGGUCUGCUGAGUCCGNN 2504 CGGACUCAGCAGACCCGGCNN 35  524-542CCGGGUCUGCUGAGUCCGCNN 2505 GCGGACUCAGCAGACCCGGNN 35  525-543CGGGUCUGCUGAGUCCGCANN 2506 UGCGGACUCAGCAGACCCGNN 35  526-544GGGUCUGCUGAGUCCGCAGNN 2507 CUGCGGACUCAGCAGACCCNN 35  574-592GUGCAGGCCCAGUUGUCACNN 2508 GUGACAACUGGGCCUGCACNN 35  575-593UGCAGGCCCAGUUGUCACCNN 2509 GGUGACAACUGGGCCUGCANN 35  576-594GCAGGCCCAGUUGUCACCCNN 2510 GGGUGACAACUGGGCCUGCNN 35  577-595CAGGCCCAGUUGUCACCCCNN 2511 GGGGUGACAACUGGGCCUGNN 35  578-596AGGCCCAGUUGUCACCCCUNN 2512 AGGGGUGACAACUGGGCCUNN 35  579-597GGCCCAGUUGUCACCCCUCNN 2513 GAGGGGUGACAACUGGGCCNN 35  580-598GCCCAGUUGUCACCCCUCCNN 2514 GGAGGGGUGACAACUGGGCNN 35  581-599CCCAGUUGUCACCCCUCCANN 2515 UGGAGGGGUGACAACUGGGNN 35  582-600CCAGUUGUCACCCCUCCAGNN 2516 CUGGAGGGGUGACAACUGGNN 35  583-601CAGUUGUCACCCCUCCAGANN 2517 UCUGGAGGGGUGACAACUGNN 35  584-602AGUUGUCACCCCUCCAGAANN 2518 UUCUGGAGGGGUGACAACUNN 35  585-603GUUGUCACCCCUCCAGAACNN 2519 GUUCUGGAGGGGUGACAACNN 35  586-604UUGUCACCCCUCCAGAACANN 2520 UGUUCUGGAGGGGUGACAANN 35  587-605UGUCACCCCUCCAGAACAUNN 2521 AUGUUCUGGAGGGGUGACANN 35  588-606GUCACCCCUCCAGAACAUCNN 2522 GAUGUUCUGGAGGGGUGACNN 35  589-607UCACCCCUCCAGAACAUCUNN 2523 AGAUGUUCUGGAGGGGUGANN 35  590-608CACCCCUCCAGAACAUCUCNN 2524 GAGAUGUUCUGGAGGGGUGNN 35  591-609ACCCCUCCAGAACAUCUCCNN 2525 GGAGAUGUUCUGGAGGGGUNN 35  592-610CCCCUCCAGAACAUCUCCCNN 2526 GGGAGAUGUUCUGGAGGGGNN 35  593-611CCCUCCAGAACAUCUCCCCNN 2527 GGGGAGAUGUUCUGGAGGGNN 35  594-612CCUCCAGAACAUCUCCCCANN 2528 UGGGGAGAUGUUCUGGAGGNN 35  595-613CUCCAGAACAUCUCCCCAUNN 2529 AUGGGGAGAUGUUCUGGAGNN 35  596-614UCCAGAACAUCUCCCCAUGNN 2530 CAUGGGGAGAUGUUCUGGANN 35  597-615CCAGAACAUCUCCCCAUGGNN 2531 CCAUGGGGAGAUGUUCUGGNN 35  598-616CAGAACAUCUCCCCAUGGANN 2532 UCCAUGGGGAGAUGUUCUGNN 35  599-617AGAACAUCUCCCCAUGGAUNN 2533 AUCCAUGGGGAGAUGUUCUNN 35  600-618GAACAUCUCCCCAUGGAUUNN 2534 AAUCCAUGGGGAGAUGUUCNN 35  601-619AACAUCUCCCCAUGGAUUCNN 2535 GAAUCCAUGGGGAGAUGUUNN 35  602-620ACAUCUCCCCAUGGAUUCUNN 2536 AGAAUCCAUGGGGAGAUGUNN 35  603-621CAUCUCCCCAUGGAUUCUGNN 2537 CAGAAUCCAUGGGGAGAUGNN 35  604-622AUCUCCCCAUGGAUUCUGGNN 2538 CCAGAAUCCAUGGGGAGAUNN 35  605-623UCUCCCCAUGGAUUCUGGCNN 2539 GCCAGAAUCCAUGGGGAGANN 35  606-624CUCCCCAUGGAUUCUGGCGNN 2540 CGCCAGAAUCCAUGGGGAGNN 35  607-625UCCCCAUGGAUUCUGGCGGNN 2541 CCGCCAGAAUCCAUGGGGANN 36  608-626CCCCAUGGAUUCUGGCGGUNN 2542 ACCGCCAGAAUCCAUGGGGNN 36  609-627CCCAUGGAUUCUGGCGGUANN 2543 UACCGCCAGAAUCCAUGGGNN 36  610-628CCAUGGAUUCUGGCGGUAUNN 2544 AUACCGCCAGAAUCCAUGGNN 36  611-629CAUGGAUUCUGGCGGUAUUNN 2545 AAUACCGCCAGAAUCCAUGNN 36  612-630AUGGAUUCUGGCGGUAUUGNN 2546 CAAUACCGCCAGAAUCCAUNN 36  613-631UGGAUUCUGGCGGUAUUGANN 2547 UCAAUACCGCCAGAAUCCANN 36  614-632GGAUUCUGGCGGUAUUGACNN 2548 GUCAAUACCGCCAGAAUCCNN 36  615-633GAUUCUGGCGGUAUUGACUNN 2549 AGUCAAUACCGCCAGAAUCNN 36  616-634AUUCUGGCGGUAUUGACUCNN 2550 GAGUCAAUACCGCCAGAAUNN 36  617-635UUCUGGCGGUAUUGACUCUNN 2551 AGAGUCAAUACCGCCAGAANN 36  618-636UCUGGCGGUAUUGACUCUUNN 2552 AAGAGUCAAUACCGCCAGANN 36  619-637CUGGCGGUAUUGACUCUUCNN 2553 GAAGAGUCAAUACCGCCAGNN 36  620-638UGGCGGUAUUGACUCUUCANN 2554 UGAAGAGUCAAUACCGCCANN 36  621-639GGCGGUAUUGACUCUUCAGNN 2555 CUGAAGAGUCAAUACCGCCNN 36  622-640GCGGUAUUGACUCUUCAGANN 2556 UCUGAAGAGUCAAUACCGCNN 36  623-641CGGUAUUGACUCUUCAGAUNN 2557 AUCUGAAGAGUCAAUACCGNN 36  624-642GGUAUUGACUCUUCAGAUUNN 2558 AAUCUGAAGAGUCAAUACCNN 36  625-643GUAUUGACUCUUCAGAUUCNN 2559 GAAUCUGAAGAGUCAAUACNN 36  626-644UAUUGACUCUUCAGAUUCANN 2560 UGAAUCUGAAGAGUCAAUANN 36  627-645AUUGACUCUUCAGAUUCAGNN 2561 CUGAAUCUGAAGAGUCAAUNN 36  628-646UUGACUCUUCAGAUUCAGANN 2562 UCUGAAUCUGAAGAGUCAANN 36  629-647UGACUCUUCAGAUUCAGAGNN 2563 CUCUGAAUCUGAAGAGUCANN 36  630-648GACUCUUCAGAUUCAGAGUNN 2564 ACUCUGAAUCUGAAGAGUCNN 36  631-649ACUCUUCAGAUUCAGAGUCNN 2565 GACUCUGAAUCUGAAGAGUNN 36  632-650CUCUUCAGAUUCAGAGUCUNN 2566 AGACUCUGAAUCUGAAGAGNN 36  633-651UCUUCAGAUUCAGAGUCUGNN 2567 CAGACUCUGAAUCUGAAGANN 36  634-652CUUCAGAUUCAGAGUCUGANN 2568 UCAGACUCUGAAUCUGAAGNN 36  635-653UUCAGAUUCAGAGUCUGAUNN 2569 AUCAGACUCUGAAUCUGAANN 36  636-654UCAGAUUCAGAGUCUGAUANN 2570 UAUCAGACUCUGAAUCUGANN 36  637-655CAGAUUCAGAGUCUGAUAUNN 2571 AUAUCAGACUCUGAAUCUGNN 36  638-656AGAUUCAGAGUCUGAUAUCNN 2572 GAUAUCAGACUCUGAAUCUNN 36  639-657GAUUCAGAGUCUGAUAUCCNN 2573 GGAUAUCAGACUCUGAAUCNN 36  640-658AUUCAGAGUCUGAUAUCCUNN 2574 AGGAUAUCAGACUCUGAAUNN 36  641-659UUCAGAGUCUGAUAUCCUGNN 2575 CAGGAUAUCAGACUCUGAANN 36  642-660UCAGAGUCUGAUAUCCUGUNN 2576 ACAGGAUAUCAGACUCUGANN 36  643-661CAGAGUCUGAUAUCCUGUUNN 2577 AACAGGAUAUCAGACUCUGNN 36  644-662AGAGUCUGAUAUCCUGUUGNN 2578 CAACAGGAUAUCAGACUCUNN 36  645-663GAGUCUGAUAUCCUGUUGGNN 2579 CCAACAGGAUAUCAGACUCNN 36  646-664AGUCUGAUAUCCUGUUGGGNN 2580 CCCAACAGGAUAUCAGACUNN 36  647-665GUCUGAUAUCCUGUUGGGCNN 2581 GCCCAACAGGAUAUCAGACNN 36  648-666UCUGAUAUCCUGUUGGGCANN 2582 UGCCCAACAGGAUAUCAGANN 36  649-667CUGAUAUCCUGUUGGGCAUNN 2583 AUGCCCAACAGGAUAUCAGNN 36  650-668UGAUAUCCUGUUGGGCAUUNN 2584 AAUGCCCAACAGGAUAUCANN 36  651-669GAUAUCCUGUUGGGCAUUCNN 2585 GAAUGCCCAACAGGAUAUCNN 36  652-670AUAUCCUGUUGGGCAUUCUNN 2586 AGAAUGCCCAACAGGAUAUNN 36  653-671UAUCCUGUUGGGCAUUCUGNN 2587 CAGAAUGCCCAACAGGAUANN 36  654-672AUCCUGUUGGGCAUUCUGGNN 2588 CCAGAAUGCCCAACAGGAUNN 36  655-673UCCUGUUGGGCAUUCUGGANN 2589 UCCAGAAUGCCCAACAGGANN 36  656-674CCUGUUGGGCAUUCUGGACNN 2590 GUCCAGAAUGCCCAACAGGNN 36  657-675CUGUUGGGCAUUCUGGACANN 2591 UGUCCAGAAUGCCCAACAGNN 36  658-676UGUUGGGCAUUCUGGACAANN 2592 UUGUCCAGAAUGCCCAACANN 36  659-677GUUGGGCAUUCUGGACAACNN 2593 GUUGUCCAGAAUGCCCAACNN 36  660-678UUGGGCAUUCUGGACAACUNN 2594 AGUUGUCCAGAAUGCCCAANN 36  661-679UGGGCAUUCUGGACAACUUNN 2595 AAGUUGUCCAGAAUGCCCANN 36  662-680GGGCAUUCUGGACAACUUGNN 2596 CAAGUUGUCCAGAAUGCCCNN 36  663-681GGCAUUCUGGACAACUUGGNN 2597 CCAAGUUGUCCAGAAUGCCNN 36  664-682GCAUUCUGGACAACUUGGANN 2598 UCCAAGUUGUCCAGAAUGCNN 36  665-683CAUUCUGGACAACUUGGACNN 2599 GUCCAAGUUGUCCAGAAUGNN 36  666-684AUUCUGGACAACUUGGACCNN 2600 GGUCCAAGUUGUCCAGAAUNN 36  667-685UUCUGGACAACUUGGACCCNN 2601 GGGUCCAAGUUGUCCAGAANN 36  668-686UCUGGACAACUUGGACCCANN 2602 UGGGUCCAAGUUGUCCAGANN 36  669-687CUGGACAACUUGGACCCAGNN 2603 CUGGGUCCAAGUUGUCCAGNN 36  670-688UGGACAACUUGGACCCAGUNN 2604 ACUGGGUCCAAGUUGUCCANN 36  671-689GGACAACUUGGACCCAGUCNN 2605 GACUGGGUCCAAGUUGUCCNN 36  672-690GACAACUUGGACCCAGUCANN 2606 UGACUGGGUCCAAGUUGUCNN 36  673-691ACAACUUGGACCCAGUCAUNN 2607 AUGACUGGGUCCAAGUUGUNN 36  674-692CAACUUGGACCCAGUCAUGNN 2608 CAUGACUGGGUCCAAGUUGNN 36  675-693AACUUGGACCCAGUCAUGUNN 2609 ACAUGACUGGGUCCAAGUUNN 36  676-694ACUUGGACCCAGUCAUGUUNN 2610 AACAUGACUGGGUCCAAGUNN 36  677-695CUUGGACCCAGUCAUGUUCNN 2611 GAACAUGACUGGGUCCAAGNN 36  678-696UUGGACCCAGUCAUGUUCUNN 2612 AGAACAUGACUGGGUCCAANN 36  679-697UGGACCCAGUCAUGUUCUUNN 2613 AAGAACAUGACUGGGUCCANN 36  680-698GGACCCAGUCAUGUUCUUCNN 2614 GAAGAACAUGACUGGGUCCNN 36  681-699GACCCAGUCAUGUUCUUCANN 2615 UGAAGAACAUGACUGGGUCNN 36  682-700ACCCAGUCAUGUUCUUCAANN 2616 UUGAAGAACAUGACUGGGUNN 36  683-701CCCAGUCAUGUUCUUCAAANN 2617 UUUGAAGAACAUGACUGGGNN 36  684-702CCAGUCAUGUUCUUCAAAUNN 2618 AUUUGAAGAACAUGACUGGNN 36  685-703CAGUCAUGUUCUUCAAAUGNN 2619 CAUUUGAAGAACAUGACUGNN 36  686-704AGUCAUGUUCUUCAAAUGCNN 2620 GCAUUUGAAGAACAUGACUNN 36  687-705GUCAUGUUCUUCAAAUGCCNN 2621 GGCAUUUGAAGAACAUGACNN 36  688-706UCAUGUUCUUCAAAUGCCCNN 2622 GGGCAUUUGAAGAACAUGANN 36  689-707CAUGUUCUUCAAAUGCCCUNN 2623 AGGGCAUUUGAAGAACAUGNN 36  690-708AUGUUCUUCAAAUGCCCUUNN 2624 AAGGGCAUUUGAAGAACAUNN 36  691-709UGUUCUUCAAAUGCCCUUCNN 2625 GAAGGGCAUUUGAAGAACANN 36  692-710GUUCUUCAAAUGCCCUUCCNN 2626 GGAAGGGCAUUUGAAGAACNN 36  693-711UUCUUCAAAUGCCCUUCCCNN 2627 GGGAAGGGCAUUUGAAGAANN 36  694-712UCUUCAAAUGCCCUUCCCCNN 2628 GGGGAAGGGCAUUUGAAGANN 36  695-713CUUCAAAUGCCCUUCCCCANN 2629 UGGGGAAGGGCAUUUGAAGNN 36  696-714UUCAAAUGCCCUUCCCCAGNN 2630 CUGGGGAAGGGCAUUUGAANN 36  697-715UCAAAUGCCCUUCCCCAGANN 2631 UCUGGGGAAGGGCAUUUGANN 36  698-716CAAAUGCCCUUCCCCAGAGNN 2632 CUCUGGGGAAGGGCAUUUGNN 36  718-736CUGCCAGCCUGGAGGAGCUNN 2633 AGCUCCUCCAGGCUGGCAGNN 36  719-737UGCCAGCCUGGAGGAGCUCNN 2634 GAGCUCCUCCAGGCUGGCANN 36  720-738GCCAGCCUGGAGGAGCUCCNN 2635 GGAGCUCCUCCAGGCUGGCNN 36  721-739CCAGCCUGGAGGAGCUCCCNN 2636 GGGAGCUCCUCCAGGCUGGNN 36  722-740CAGCCUGGAGGAGCUCCCANN 2637 UGGGAGCUCCUCCAGGCUGNN 36  723-741AGCCUGGAGGAGCUCCCAGNN 2638 CUGGGAGCUCCUCCAGGCUNN 36  724-742GCCUGGAGGAGCUCCCAGANN 2639 UCUGGGAGCUCCUCCAGGCNN 36  725-743CCUGGAGGAGCUCCCAGAGNN 2640 CUCUGGGAGCUCCUCCAGGNN 36  726-744CUGGAGGAGCUCCCAGAGGNN 2641 CCUCUGGGAGCUCCUCCAGNN 37  727-745UGGAGGAGCUCCCAGAGGUNN 2642 ACCUCUGGGAGCUCCUCCANN 37  728-746GGAGGAGCUCCCAGAGGUCNN 2643 GACCUCUGGGAGCUCCUCCNN 37  729-747GAGGAGCUCCCAGAGGUCUNN 2644 AGACCUCUGGGAGCUCCUCNN 37  730-748AGGAGCUCCCAGAGGUCUANN 2645 UAGACCUCUGGGAGCUCCUNN 37  731-749GGAGCUCCCAGAGGUCUACNN 2646 GUAGACCUCUGGGAGCUCCNN 37  732-750GAGCUCCCAGAGGUCUACCNN 2647 GGUAGACCUCUGGGAGCUCNN 37  733-751AGCUCCCAGAGGUCUACCCNN 2648 GGGUAGACCUCUGGGAGCUNN 37  734-752GCUCCCAGAGGUCUACCCANN 2649 UGGGUAGACCUCUGGGAGCNN 37  735-753CUCCCAGAGGUCUACCCAGNN 2650 CUGGGUAGACCUCUGGGAGNN 37  736-754UCCCAGAGGUCUACCCAGANN 2651 UCUGGGUAGACCUCUGGGANN 37  737-755CCCAGAGGUCUACCCAGAANN 2652 UUCUGGGUAGACCUCUGGGNN 37  738-756CCAGAGGUCUACCCAGAAGNN 2653 CUUCUGGGUAGACCUCUGGNN 37  739-757CAGAGGUCUACCCAGAAGGNN 2654 CCUUCUGGGUAGACCUCUGNN 37  740-758AGAGGUCUACCCAGAAGGANN 2655 UCCUUCUGGGUAGACCUCUNN 37  741-759GAGGUCUACCCAGAAGGACNN 2656 GUCCUUCUGGGUAGACCUCNN 37  742-760AGGUCUACCCAGAAGGACCNN 2657 GGUCCUUCUGGGUAGACCUNN 37  743-761GGUCUACCCAGAAGGACCCNN 2658 GGGUCCUUCUGGGUAGACCNN 37  744-762GUCUACCCAGAAGGACCCANN 2659 UGGGUCCUUCUGGGUAGACNN 37  745-763UCUACCCAGAAGGACCCAGNN 2660 CUGGGUCCUUCUGGGUAGANN 37  746-764CUACCCAGAAGGACCCAGUNN 2661 ACUGGGUCCUUCUGGGUAGNN 37  747-765UACCCAGAAGGACCCAGUUNN 2662 AACUGGGUCCUUCUGGGUANN 37  748-766ACCCAGAAGGACCCAGUUCNN 2663 GAACUGGGUCCUUCUGGGUNN 37  749-767CCCAGAAGGACCCAGUUCCNN 2664 GGAACUGGGUCCUUCUGGGNN 37  750-768CCAGAAGGACCCAGUUCCUNN 2665 AGGAACUGGGUCCUUCUGGNN 37  751-769CAGAAGGACCCAGUUCCUUNN 2666 AAGGAACUGGGUCCUUCUGNN 37  752-770AGAAGGACCCAGUUCCUUANN 2667 UAAGGAACUGGGUCCUUCUNN 37  753-771GAAGGACCCAGUUCCUUACNN 2668 GUAAGGAACUGGGUCCUUCNN 37  754-772AAGGACCCAGUUCCUUACCNN 2669 GGUAAGGAACUGGGUCCUUNN 37  755-773AGGACCCAGUUCCUUACCANN 2670 UGGUAAGGAACUGGGUCCUNN 37  756-774GGACCCAGUUCCUUACCAGNN 2671 CUGGUAAGGAACUGGGUCCNN 37  757-775GACCCAGUUCCUUACCAGCNN 2672 GCUGGUAAGGAACUGGGUCNN 37  758-776ACCCAGUUCCUUACCAGCCNN 2673 GGCUGGUAAGGAACUGGGUNN 37  759-777CCCAGUUCCUUACCAGCCUNN 2674 AGGCUGGUAAGGAACUGGGNN 37  760-778CCAGUUCCUUACCAGCCUCNN 2675 GAGGCUGGUAAGGAACUGGNN 37  761-779CAGUUCCUUACCAGCCUCCNN 2676 GGAGGCUGGUAAGGAACUGNN 37  762-780AGUUCCUUACCAGCCUCCCNN 2677 GGGAGGCUGGUAAGGAACUNN 37  763-781GUUCCUUACCAGCCUCCCUNN 2678 AGGGAGGCUGGUAAGGAACNN 37  764-782UUCCUUACCAGCCUCCCUUNN 2679 AAGGGAGGCUGGUAAGGAANN 37  765-783UCCUUACCAGCCUCCCUUUNN 2680 AAAGGGAGGCUGGUAAGGANN 37  766-784CCUUACCAGCCUCCCUUUCNN 2681 GAAAGGGAGGCUGGUAAGGNN 37  767-785CUUACCAGCCUCCCUUUCUNN 2682 AGAAAGGGAGGCUGGUAAGNN 37  768-786UUACCAGCCUCCCUUUCUCNN 2683 GAGAAAGGGAGGCUGGUAANN 37  769-787UACCAGCCUCCCUUUCUCUNN 2684 AGAGAAAGGGAGGCUGGUANN 37  770-788ACCAGCCUCCCUUUCUCUGNN 2685 CAGAGAAAGGGAGGCUGGUNN 37  771-789CCAGCCUCCCUUUCUCUGUNN 2686 ACAGAGAAAGGGAGGCUGGNN 37  772-790CAGCCUCCCUUUCUCUGUCNN 2687 GACAGAGAAAGGGAGGCUGNN 37  773-791AGCCUCCCUUUCUCUGUCANN 2688 UGACAGAGAAAGGGAGGCUNN 37  774-792GCCUCCCUUUCUCUGUCAGNN 2689 CUGACAGAGAAAGGGAGGCNN 37  775-793CCUCCCUUUCUCUGUCAGUNN 2690 ACUGACAGAGAAAGGGAGGNN 37  776-794CUCCCUUUCUCUGUCAGUGNN 2691 CACUGACAGAGAAAGGGAGNN 37  777-795UCCCUUUCUCUGUCAGUGGNN 2692 CCACUGACAGAGAAAGGGANN 37  778-796CCCUUUCUCUGUCAGUGGGNN 2693 CCCACUGACAGAGAAAGGGNN 37  779-797CCUUUCUCUGUCAGUGGGGNN 2694 CCCCACUGACAGAGAAAGGNN 37  780-798CUUUCUCUGUCAGUGGGGANN 2695 UCCCCACUGACAGAGAAAGNN 37  781-799UUUCUCUGUCAGUGGGGACNN 2696 GUCCCCACUGACAGAGAAANN 37  782-800UUCUCUGUCAGUGGGGACGNN 2697 CGUCCCCACUGACAGAGAANN 37  783-801UCUCUGUCAGUGGGGACGUNN 2698 ACGUCCCCACUGACAGAGANN 37  784-802CUCUGUCAGUGGGGACGUCNN 2699 GACGUCCCCACUGACAGAGNN 37  785-803UCUGUCAGUGGGGACGUCANN 2700 UGACGUCCCCACUGACAGANN 37  786-804CUGUCAGUGGGGACGUCAUNN 2701 AUGACGUCCCCACUGACAGNN 37  787-805UGUCAGUGGGGACGUCAUCNN 2702 GAUGACGUCCCCACUGACANN 37  788-806GUCAGUGGGGACGUCAUCANN 2703 UGAUGACGUCCCCACUGACNN 37  789-807UCAGUGGGGACGUCAUCAGNN 2704 CUGAUGACGUCCCCACUGANN 37  790-808CAGUGGGGACGUCAUCAGCNN 2705 GCUGAUGACGUCCCCACUGNN 37  791-809AGUGGGGACGUCAUCAGCCNN 2706 GGCUGAUGACGUCCCCACUNN 37  792-810GUGGGGACGUCAUCAGCCANN 2707 UGGCUGAUGACGUCCCCACNN 37  793-811UGGGGACGUCAUCAGCCAANN 2708 UUGGCUGAUGACGUCCCCANN 37  794-812GGGGACGUCAUCAGCCAAGNN 2709 CUUGGCUGAUGACGUCCCCNN 37  795-813GGGACGUCAUCAGCCAAGCNN 2710 GCUUGGCUGAUGACGUCCCNN 37  796-814GGACGUCAUCAGCCAAGCUNN 2711 AGCUUGGCUGAUGACGUCCNN 37  797-815GACGUCAUCAGCCAAGCUGNN 2712 CAGCUUGGCUGAUGACGUCNN 37  798-816ACGUCAUCAGCCAAGCUGGNN 2713 CCAGCUUGGCUGAUGACGUNN 37  799-817CGUCAUCAGCCAAGCUGGANN 2714 UCCAGCUUGGCUGAUGACGNN 37  800-818GUCAUCAGCCAAGCUGGAANN 2715 UUCCAGCUUGGCUGAUGACNN 37  801-819UCAUCAGCCAAGCUGGAAGNN 2716 CUUCCAGCUUGGCUGAUGANN 37  802-820CAUCAGCCAAGCUGGAAGCNN 2717 GCUUCCAGCUUGGCUGAUGNN 37  803-821AUCAGCCAAGCUGGAAGCCNN 2718 GGCUUCCAGCUUGGCUGAUNN 37  804-822UCAGCCAAGCUGGAAGCCANN 2719 UGGCUUCCAGCUUGGCUGANN 37  805-823CAGCCAAGCUGGAAGCCAUNN 2720 AUGGCUUCCAGCUUGGCUGNN 37  806-824AGCCAAGCUGGAAGCCAUUNN 2721 AAUGGCUUCCAGCUUGGCUNN 37  807-825GCCAAGCUGGAAGCCAUUANN 2722 UAAUGGCUUCCAGCUUGGCNN 37  808-826CCAAGCUGGAAGCCAUUAANN 2723 UUAAUGGCUUCCAGCUUGGNN 37  809-827CAAGCUGGAAGCCAUUAAUNN 2724 AUUAAUGGCUUCCAGCUUGNN 37  810-828AAGCUGGAAGCCAUUAAUGNN 2725 CAUUAAUGGCUUCCAGCUUNN 37  811-829AGCUGGAAGCCAUUAAUGANN 2726 UCAUUAAUGGCUUCCAGCUNN 37  812-830GCUGGAAGCCAUUAAUGAANN 2727 UUCAUUAAUGGCUUCCAGCNN 37  813-831CUGGAAGCCAUUAAUGAACNN 2728 GUUCAUUAAUGGCUUCCAGNN 37  814-832UGGAAGCCAUUAAUGAACUNN 2729 AGUUCAUUAAUGGCUUCCANN 37  815-833GGAAGCCAUUAAUGAACUANN 2730 UAGUUCAUUAAUGGCUUCCNN 37  816-834GAAGCCAUUAAUGAACUAANN 2731 UUAGUUCAUUAAUGGCUUCNN 37  817-835AAGCCAUUAAUGAACUAAUNN 2732 AUUAGUUCAUUAAUGGCUUNN 37  818-836AGCCAUUAAUGAACUAAUUNN 2733 AAUUAGUUCAUUAAUGGCUNN 37  819-837GCCAUUAAUGAACUAAUUCNN 2734 GAAUUAGUUCAUUAAUGGCNN 37  820-838CCAUUAAUGAACUAAUUCGNN 2735 CGAAUUAGUUCAUUAAUGGNN 37  821-839CAUUAAUGAACUAAUUCGUNN 2736 ACGAAUUAGUUCAUUAAUGNN 37  822-840AUUAAUGAACUAAUUCGUUNN 2737 AACGAAUUAGUUCAUUAAUNN 37  823-841UUAAUGAACUAAUUCGUUUNN 2738 AAACGAAUUAGUUCAUUAANN 37  824-842UAAUGAACUAAUUCGUUUUNN 2739 AAAACGAAUUAGUUCAUUANN 37  825-843AAUGAACUAAUUCGUUUUGNN 2740 CAAAACGAAUUAGUUCAUUNN 37  826-844AUGAACUAAUUCGUUUUGANN 2741 UCAAAACGAAUUAGUUCAUNN 38  827-845UGAACUAAUUCGUUUUGACNN 2742 GUCAAAACGAAUUAGUUCANN 38  828-846GAACUAAUUCGUUUUGACCNN 2743 GGUCAAAACGAAUUAGUUCNN 38  829-847AACUAAUUCGUUUUGACCANN 2744 UGGUCAAAACGAAUUAGUUNN 38  830-848ACUAAUUCGUUUUGACCACNN 2745 GUGGUCAAAACGAAUUAGUNN 38  831-849CUAAUUCGUUUUGACCACANN 2746 UGUGGUCAAAACGAAUUAGNN 38  832-850UAAUUCGUUUUGACCACAUNN 2747 AUGUGGUCAAAACGAAUUANN 38  833-851AAUUCGUUUUGACCACAUANN 2748 UAUGUGGUCAAAACGAAUUNN 38  834-852AUUCGUUUUGACCACAUAUNN 2749 AUAUGUGGUCAAAACGAAUNN 38  835-853UUCGUUUUGACCACAUAUANN 2750 UAUAUGUGGUCAAAACGAANN 38  836-854UCGUUUUGACCACAUAUAUNN 2751 AUAUAUGUGGUCAAAACGANN 38  837-855CGUUUUGACCACAUAUAUANN 2752 UAUAUAUGUGGUCAAAACGNN 38  838-856GUUUUGACCACAUAUAUACNN 2753 GUAUAUAUGUGGUCAAAACNN 38  839-857UUUUGACCACAUAUAUACCNN 2754 GGUAUAUAUGUGGUCAAAANN 38  840-858UUUGACCACAUAUAUACCANN 2755 UGGUAUAUAUGUGGUCAAANN 38  841-859UUGACCACAUAUAUACCAANN 2756 UUGGUAUAUAUGUGGUCAANN 38  842-860UGACCACAUAUAUACCAAGNN 2757 CUUGGUAUAUAUGUGGUCANN 38  843-861GACCACAUAUAUACCAAGCNN 2758 GCUUGGUAUAUAUGUGGUCNN 38  844-862ACCACAUAUAUACCAAGCCNN 2759 GGCUUGGUAUAUAUGUGGUNN 38  845-863CCACAUAUAUACCAAGCCCNN 2760 GGGCUUGGUAUAUAUGUGGNN 38  846-864CACAUAUAUACCAAGCCCCNN 2761 GGGGCUUGGUAUAUAUGUGNN 38  847-865ACAUAUAUACCAAGCCCCUNN 2762 AGGGGCUUGGUAUAUAUGUNN 38  867-885GUCUUAGAGAUACCCUCUGNN 2763 CAGAGGGUAUCUCUAAGACNN 38  868-886UCUUAGAGAUACCCUCUGANN 2764 UCAGAGGGUAUCUCUAAGANN 38  869-887CUUAGAGAUACCCUCUGAGNN 2765 CUCAGAGGGUAUCUCUAAGNN 38  870-888UUAGAGAUACCCUCUGAGANN 2766 UCUCAGAGGGUAUCUCUAANN 38  871-889UAGAGAUACCCUCUGAGACNN 2767 GUCUCAGAGGGUAUCUCUANN 38  872-890AGAGAUACCCUCUGAGACANN 2768 UGUCUCAGAGGGUAUCUCUNN 38  873-891GAGAUACCCUCUGAGACAGNN 2769 CUGUCUCAGAGGGUAUCUCNN 38  874-892AGAUACCCUCUGAGACAGANN 2770 UCUGUCUCAGAGGGUAUCUNN 38  875-893GAUACCCUCUGAGACAGAGNN 2771 CUCUGUCUCAGAGGGUAUCNN 38  876-894AUACCCUCUGAGACAGAGANN 2772 UCUCUGUCUCAGAGGGUAUNN 38  877-895UACCCUCUGAGACAGAGAGNN 2773 CUCUCUGUCUCAGAGGGUANN 38  878-896ACCCUCUGAGACAGAGAGCNN 2774 GCUCUCUGUCUCAGAGGGUNN 38  879-897CCCUCUGAGACAGAGAGCCNN 2775 GGCUCUCUGUCUCAGAGGGNN 38  880-898CCUCUGAGACAGAGAGCCANN 2776 UGGCUCUCUGUCUCAGAGGNN 38  881-899CUCUGAGACAGAGAGCCAANN 2777 UUGGCUCUCUGUCUCAGAGNN 38  882-900UCUGAGACAGAGAGCCAAGNN 2778 CUUGGCUCUCUGUCUCAGANN 38  883-901CUGAGACAGAGAGCCAAGCNN 2779 GCUUGGCUCUCUGUCUCAGNN 38  884-902UGAGACAGAGAGCCAAGCUNN 2780 AGCUUGGCUCUCUGUCUCANN 38  885-903GAGACAGAGAGCCAAGCUANN 2781 UAGCUUGGCUCUCUGUCUCNN 38  886-904AGACAGAGAGCCAAGCUAANN 2782 UUAGCUUGGCUCUCUGUCUNN 38  887-905GACAGAGAGCCAAGCUAAUNN 2783 AUUAGCUUGGCUCUCUGUCNN 38  888-906ACAGAGAGCCAAGCUAAUGNN 2784 CAUUAGCUUGGCUCUCUGUNN 38  889-907CAGAGAGCCAAGCUAAUGUNN 2785 ACAUUAGCUUGGCUCUCUGNN 38  890-908AGAGAGCCAAGCUAAUGUGNN 2786 CACAUUAGCUUGGCUCUCUNN 38  891-909GAGAGCCAAGCUAAUGUGGNN 2787 CCACAUUAGCUUGGCUCUCNN 38  892-910AGAGCCAAGCUAAUGUGGUNN 2788 ACCACAUUAGCUUGGCUCUNN 38  893-911GAGCCAAGCUAAUGUGGUANN 2789 UACCACAUUAGCUUGGCUCNN 38  894-912AGCCAAGCUAAUGUGGUAGNN 2790 CUACCACAUUAGCUUGGCUNN 38  895-913GCCAAGCUAAUGUGGUAGUNN 2791 ACUACCACAUUAGCUUGGCNN 38  896-914CCAAGCUAAUGUGGUAGUGNN 2792 CACUACCACAUUAGCUUGGNN 38  897-915CAAGCUAAUGUGGUAGUGANN 2793 UCACUACCACAUUAGCUUGNN 38  898-916AAGCUAAUGUGGUAGUGAANN 2794 UUCACUACCACAUUAGCUUNN 38  899-917AGCUAAUGUGGUAGUGAAANN 2795 UUUCACUACCACAUUAGCUNN 38  900-918GCUAAUGUGGUAGUGAAAANN 2796 UUUUCACUACCACAUUAGCNN 38  901-919CUAAUGUGGUAGUGAAAAUNN 2797 AUUUUCACUACCACAUUAGNN 38  902-920UAAUGUGGUAGUGAAAAUCNN 2798 GAUUUUCACUACCACAUUANN 38  903-921AAUGUGGUAGUGAAAAUCGNN 2799 CGAUUUUCACUACCACAUUNN 38  904-922AUGUGGUAGUGAAAAUCGANN 2800 UCGAUUUUCACUACCACAUNN 38  905-923UGUGGUAGUGAAAAUCGAGNN 2801 CUCGAUUUUCACUACCACANN 38  906-924GUGGUAGUGAAAAUCGAGGNN 2802 CCUCGAUUUUCACUACCACNN 38  907-925UGGUAGUGAAAAUCGAGGANN 2803 UCCUCGAUUUUCACUACCANN 38  908-926GGUAGUGAAAAUCGAGGAANN 2804 UUCCUCGAUUUUCACUACCNN 38  909-927GUAGUGAAAAUCGAGGAAGNN 2805 CUUCCUCGAUUUUCACUACNN 38  910-928UAGUGAAAAUCGAGGAAGCNN 2806 GCUUCCUCGAUUUUCACUANN 38  911-929AGUGAAAAUCGAGGAAGCANN 2807 UGCUUCCUCGAUUUUCACUNN 38  912-930GUGAAAAUCGAGGAAGCACNN 2808 GUGCUUCCUCGAUUUUCACNN 38  913-931UGAAAAUCGAGGAAGCACCNN 2809 GGUGCUUCCUCGAUUUUCANN 38  914-932GAAAAUCGAGGAAGCACCUNN 2810 AGGUGCUUCCUCGAUUUUCNN 38  915-933AAAAUCGAGGAAGCACCUCNN 2811 GAGGUGCUUCCUCGAUUUUNN 38  916-934AAAUCGAGGAAGCACCUCUNN 2812 AGAGGUGCUUCCUCGAUUUNN 38  917-935AAUCGAGGAAGCACCUCUCNN 2813 GAGAGGUGCUUCCUCGAUUNN 38  918-936AUCGAGGAAGCACCUCUCANN 2814 UGAGAGGUGCUUCCUCGAUNN 38  919-937UCGAGGAAGCACCUCUCAGNN 2815 CUGAGAGGUGCUUCCUCGANN 38  920-938CGAGGAAGCACCUCUCAGCNN 2816 GCUGAGAGGUGCUUCCUCGNN 38  921-939GAGGAAGCACCUCUCAGCCNN 2817 GGCUGAGAGGUGCUUCCUCNN 38  922-940AGGAAGCACCUCUCAGCCCNN 2818 GGGCUGAGAGGUGCUUCCUNN 38  923-941GGAAGCACCUCUCAGCCCCNN 2819 GGGGCUGAGAGGUGCUUCCNN 38  924-942GAAGCACCUCUCAGCCCCUNN 2820 AGGGGCUGAGAGGUGCUUCNN 38  925-943AAGCACCUCUCAGCCCCUCNN 2821 GAGGGGCUGAGAGGUGCUUNN 38  926-944AGCACCUCUCAGCCCCUCANN 2822 UGAGGGGCUGAGAGGUGCUNN 38  927-945GCACCUCUCAGCCCCUCAGNN 2823 CUGAGGGGCUGAGAGGUGCNN 38  928-946CACCUCUCAGCCCCUCAGANN 2824 UCUGAGGGGCUGAGAGGUGNN 38  929-947ACCUCUCAGCCCCUCAGAGNN 2825 CUCUGAGGGGCUGAGAGGUNN 38  930-948CCUCUCAGCCCCUCAGAGANN 2826 UCUCUGAGGGGCUGAGAGGNN 38  931-949CUCUCAGCCCCUCAGAGAANN 2827 UUCUCUGAGGGGCUGAGAGNN 38  932-950UCUCAGCCCCUCAGAGAAUNN 2828 AUUCUCUGAGGGGCUGAGANN 38  933-951CUCAGCCCCUCAGAGAAUGNN 2829 CAUUCUCUGAGGGGCUGAGNN 38  934-952UCAGCCCCUCAGAGAAUGANN 2830 UCAUUCUCUGAGGGGCUGANN 38  935-953CAGCCCCUCAGAGAAUGAUNN 2831 AUCAUUCUCUGAGGGGCUGNN 38  936-954AGCCCCUCAGAGAAUGAUCNN 2832 GAUCAUUCUCUGAGGGGCUNN 38  937-955GCCCCUCAGAGAAUGAUCANN 2833 UGAUCAUUCUCUGAGGGGCNN 38  938-956CCCCUCAGAGAAUGAUCACNN 2834 GUGAUCAUUCUCUGAGGGGNN 38  939-957CCCUCAGAGAAUGAUCACCNN 2835 GGUGAUCAUUCUCUGAGGGNN 38  940-958CCUCAGAGAAUGAUCACCCNN 2836 GGGUGAUCAUUCUCUGAGGNN 38  941-959CUCAGAGAAUGAUCACCCUNN 2837 AGGGUGAUCAUUCUCUGAGNN 38  942-960UCAGAGAAUGAUCACCCUGNN 2838 CAGGGUGAUCAUUCUCUGANN 38  943-961CAGAGAAUGAUCACCCUGANN 2839 UCAGGGUGAUCAUUCUCUGNN 38  944-962AGAGAAUGAUCACCCUGAANN 2840 UUCAGGGUGAUCAUUCUCUNN 38  945-963GAGAAUGAUCACCCUGAAUNN 2841 AUUCAGGGUGAUCAUUCUCNN 39  946-964AGAAUGAUCACCCUGAAUUNN 2842 AAUUCAGGGUGAUCAUUCUNN 39  947-965GAAUGAUCACCCUGAAUUCNN 2843 GAAUUCAGGGUGAUCAUUCNN 39  948-966AAUGAUCACCCUGAAUUCANN 2844 UGAAUUCAGGGUGAUCAUUNN 39  949-967AUGAUCACCCUGAAUUCAUNN 2845 AUGAAUUCAGGGUGAUCAUNN 39  950-968UGAUCACCCUGAAUUCAUUNN 2846 AAUGAAUUCAGGGUGAUCANN 39  951-969GAUCACCCUGAAUUCAUUGNN 2847 CAAUGAAUUCAGGGUGAUCNN 39  952-970AUCACCCUGAAUUCAUUGUNN 2848 ACAAUGAAUUCAGGGUGAUNN 39  953-971UCACCCUGAAUUCAUUGUCNN 2849 GACAAUGAAUUCAGGGUGANN 39  954-972CACCCUGAAUUCAUUGUCUNN 2850 AGACAAUGAAUUCAGGGUGNN 39  955-973ACCCUGAAUUCAUUGUCUCNN 2851 GAGACAAUGAAUUCAGGGUNN 39  956-974CCCUGAAUUCAUUGUCUCANN 2852 UGAGACAAUGAAUUCAGGGNN 39  957-975CCUGAAUUCAUUGUCUCAGNN 2853 CUGAGACAAUGAAUUCAGGNN 39  958-976CUGAAUUCAUUGUCUCAGUNN 2854 ACUGAGACAAUGAAUUCAGNN 39  959-977UGAAUUCAUUGUCUCAGUGNN 2855 CACUGAGACAAUGAAUUCANN 39  960-978GAAUUCAUUGUCUCAGUGANN 2856 UCACUGAGACAAUGAAUUCNN 39  961-979AAUUCAUUGUCUCAGUGAANN 2857 UUCACUGAGACAAUGAAUUNN 39  962-980AUUCAUUGUCUCAGUGAAGNN 2858 CUUCACUGAGACAAUGAAUNN 39  963-981UUCAUUGUCUCAGUGAAGGNN 2859 CCUUCACUGAGACAAUGAANN 39  964-982UCAUUGUCUCAGUGAAGGANN 2860 UCCUUCACUGAGACAAUGANN 39  965-983CAUUGUCUCAGUGAAGGAANN 2861 UUCCUUCACUGAGACAAUGNN 39  966-984AUUGUCUCAGUGAAGGAAGNN 2862 CUUCCUUCACUGAGACAAUNN 39  967-985UUGUCUCAGUGAAGGAAGANN 2863 UCUUCCUUCACUGAGACAANN 39  968-986UGUCUCAGUGAAGGAAGAANN 2864 UUCUUCCUUCACUGAGACANN 39  969-987GUCUCAGUGAAGGAAGAACNN 2865 GUUCUUCCUUCACUGAGACNN 39  970-988UCUCAGUGAAGGAAGAACCNN 2866 GGUUCUUCCUUCACUGAGANN 39  971-989CUCAGUGAAGGAAGAACCUNN 2867 AGGUUCUUCCUUCACUGAGNN 39  972-990UCAGUGAAGGAAGAACCUGNN 2868 CAGGUUCUUCCUUCACUGANN 39  973-991CAGUGAAGGAAGAACCUGUNN 2869 ACAGGUUCUUCCUUCACUGNN 39  974-992AGUGAAGGAAGAACCUGUANN 2870 UACAGGUUCUUCCUUCACUNN 39  975-993GUGAAGGAAGAACCUGUAGNN 2871 CUACAGGUUCUUCCUUCACNN 39  976-994UGAAGGAAGAACCUGUAGANN 2872 UCUACAGGUUCUUCCUUCANN 39  977-995GAAGGAAGAACCUGUAGAANN 2873 UUCUACAGGUUCUUCCUUCNN 39  978-996AAGGAAGAACCUGUAGAAGNN 2874 CUUCUACAGGUUCUUCCUUNN 39  979-997AGGAAGAACCUGUAGAAGANN 2875 UCUUCUACAGGUUCUUCCUNN 39  980-998GGAAGAACCUGUAGAAGAUNN 2876 AUCUUCUACAGGUUCUUCCNN 39  981-999GAAGAACCUGUAGAAGAUGNN 2877 CAUCUUCUACAGGUUCUUCNN 39  982-1000AAGAACCUGUAGAAGAUGANN 2878 UCAUCUUCUACAGGUUCUUNN 39  983-1001AGAACCUGUAGAAGAUGACNN 2879 GUCAUCUUCUACAGGUUCUNN 39  984-1002GAACCUGUAGAAGAUGACCNN 2880 GGUCAUCUUCUACAGGUUCNN 39  985-1003AACCUGUAGAAGAUGACCUNN 2881 AGGUCAUCUUCUACAGGUUNN 39  986-1004ACCUGUAGAAGAUGACCUCNN 2882 GAGGUCAUCUUCUACAGGUNN 39

TABLE 13Sequences of dsRNA targeting both mouse and rhesus monkey XBP-1.*Target refers location of target sequence in NM_013842 (Mus musculis XPB1 mRNA).Sense and antisense sequences are described with optional dinucleotide (NN) overhangs.SEQ ID SEQ ID *Target sense (5′-3′) NO antisense (5′-3′) NO  369-387AGAAAACUCACGGCCUUGUNN 3942 ACAAGGCCGUGAGUUUUCUNN 4042  237-255AACUGAAAAACAGAGUAGCNN 3943 GCUACUCUGUUUUUCAGUUNN 4043  491-509GGGUCUGCUGAGUCCGCAGNN 3944 CUGCGGACUCAGCAGACCCNN 4044  917-935AUCACCCUGAAUUCAUUGUNN 3945 ACAAUGAAUUCAGGGUGAUNN 4045  923-941CUGAAUUCAUUGUCUCAGUNN 3946 ACUGAGACAAUGAAUUCAGNN 4046  702-720CCCAGAGGUCUACCCAGAANN 3947 UUCUGGGUAGACCUCUGGGNN 4047  926-944AAUUCAUUGUCUCAGUGAANN 3948 UUCACUGAGACAAUGAAUUNN 4048  391-409UGAGAACCAGGAGUUAAGANN 3949 UCUUAACUCCUGGUUCUCANN 4049  775-793AAGCUGGAAGCCAUUAAUGNN 3950 CAUUAAUGGCUUCCAGCUUNN 4050 1150-1168CCCCAGCUGAUUAGUGUCUNN 3951 AGACACUAAUCAGCUGGGGNN 4051  776-794AGCUGGAAGCCAUUAAUGANN 3952 UCAUUAAUGGCUUCCAGCUNN 4052  921-939CCCUGAAUUCAUUGUCUCANN 3953 UGAGACAAUGAAUUCAGGGNN 4053  777-795GCUGGAAGCCAUUAAUGAANN 3954 UUCAUUAAUGGCUUCCAGCNN 4054  539-557GUGCAGGCCCAGUUGUCACNN 3955 GUGACAACUGGGCCUGCACNN 4055  731-749CCUUACCAGCCUCCCUUUCNN 3956 GAAAGGGAGGCUGGUAAGGNN 4056  924-942UGAAUUCAUUGUCUCAGUGNN 3957 CACUGAGACAAUGAAUUCANN 4057 1151-1169CCCAGCUGAUUAGUGUCUANN 3958 UAGACACUAAUCAGCUGGGNN 4058 1152-1170CCAGCUGAUUAGUGUCUAANN 3959 UUAGACACUAAUCAGCUGGNN 4059 1718-1736ACUAUGUAAAUGCUUGAUGNN 3960 CAUCAAGCAUUUACAUAGUNN 4060  368-386GAGAAAACUCACGGCCUUGNN 3961 CAAGGCCGUGAGUUUUCUCNN 4061  489-507CCGGGUCUGCUGAGUCCGCNN 3962 GCGGACUCAGCAGACCCGGNN 4062  238-256ACUGAAAAACAGAGUAGCANN 3963 UGCUACUCUGUUUUUCAGUNN 4063  240-258UGAAAAACAGAGUAGCAGCNN 3964 GCUGCUACUCUGUUUUUCANN 4064  390-408UUGAGAACCAGGAGUUAAGNN 3965 CUUAACUCCUGGUUCUCAANN 4065  487-505GGCCGGGUCUGCUGAGUCCNN 3966 GGACUCAGCAGACCCGGCCNN 4066  741-759CUCCCUUUCUCUGUCAGUGNN 3967 CACUGACAGAGAAAGGGAGNN 4067  918-936UCACCCUGAAUUCAUUGUCNN 3968 GACAAUGAAUUCAGGGUGANN 4068  919-937CACCCUGAAUUCAUUGUCUNN 3969 AGACAAUGAAUUCAGGGUGNN 4069 1130-1148CUUUUGCCAAUGAACUUUUNN 3970 AAAAGUUCAUUGGCAAAAGNN 4070 1712-1730AAAUUUACUAUGUAAAUGCNN 3971 GCAUUUACAUAGUAAAUUUNN 4071 1714-1732AUUUACUAUGUAAAUGCUUNN 3972 AAGCAUUUACAUAGUAAAUNN 4072 1717-1735UACUAUGUAAAUGCUUGAUNN 3973 AUCAAGCAUUUACAUAGUANN 4073 1719-1737CUAUGUAAAUGCUUGAUGGNN 3974 CCAUCAAGCAUUUACAUAGNN 4074 1775-1793CCAUUUAUUUAAAACUACCNN 3975 GGUAGUUUUAAAUAAAUGGNN 4075 1776-1794CAUUUAUUUAAAACUACCCNN 3976 GGGUAGUUUUAAAUAAAUGNN 4076  239-257CUGAAAAACAGAGUAGCAGNN 3977 CUGCUACUCUGUUUUUCAGNN 4077  347-365CUAGAAAAUCAGCUUUUACNN 3978 GUAAAAGCUGAUUUUCUAGNN 4078  348-366UAGAAAAUCAGCUUUUACGNN 3979 CGUAAAAGCUGAUUUUCUANN 4079  485-503GUGGCCGGGUCUGCUGAGUNN 3980 ACUCAGCAGACCCGGCCACNN 4080  486-504UGGCCGGGUCUGCUGAGUCNN 3981 GACUCAGCAGACCCGGCCANN 4081  488-506GCCGGGUCUGCUGAGUCCGNN 3982 CGGACUCAGCAGACCCGGCNN 4082  540-558UGCAGGCCCAGUUGUCACCNN 3983 GGUGACAACUGGGCCUGCANN 4083  703-721CCAGAGGUCUACCCAGAAGNN 3984 CUUCUGGGUAGACCUCUGGNN 4084  705-723AGAGGUCUACCCAGAAGGANN 3985 UCCUUCUGGGUAGACCUCUNN 4085  730-748UCCUUACCAGCCUCCCUUUNN 3986 AAAGGGAGGCUGGUAAGGANN 4086  742-760UCCCUUUCUCUGUCAGUGGNN 3987 CCACUGACAGAGAAAGGGANN 4087  744-762CCUUUCUCUGUCAGUGGGGNN 3988 CCCCACUGACAGAGAAAGGNN 4088  767-785CAUCAGCCAAGCUGGAAGCNN 3989 GCUUCCAGCUUGGCUGAUGNN 4089  771-789AGCCAAGCUGGAAGCCAUUNN 3990 AAUGGCUUCCAGCUUGGCUNN 4090  916-934GAUCACCCUGAAUUCAUUGNN 3991 CAAUGAAUUCAGGGUGAUCNN 4091  920-938ACCCUGAAUUCAUUGUCUCNN 3992 GAGACAAUGAAUUCAGGGUNN 4092  922-940CCUGAAUUCAUUGUCUCAGNN 3993 CUGAGACAAUGAAUUCAGGNN 4093  925-943GAAUUCAUUGUCUCAGUGANN 3994 UCACUGAGACAAUGAAUUCNN 4094 1720-1738UAUGUAAAUGCUUGAUGGANN 3995 UCCAUCAAGCAUUUACAUANN 4095  232-250GAGGAAACUGAAAAACAGANN 3996 UCUGUUUUUCAGUUUCCUCNN 4096  236-254AAACUGAAAAACAGAGUAGNN 3997 CUACUCUGUUUUUCAGUUUNN 4097  728-746GUUCCUUACCAGCCUCCCUNN 3998 AGGGAGGCUGGUAAGGAACNN 4098  729-747UUCCUUACCAGCCUCCCUUNN 3999 AAGGGAGGCUGGUAAGGAANN 4099  745-763CUUUCUCUGUCAGUGGGGANN 4000 UCCCCACUGACAGAGAAAGNN 4100  766-784UCAUCAGCCAAGCUGGAAGNN 4001 CUUCCAGCUUGGCUGAUGANN 4101  927-945AUUCAUUGUCUCAGUGAAGNN 4002 CUUCACUGAGACAAUGAAUNN 4102  234-252GGAAACUGAAAAACAGAGUNN 4003 ACUCUGUUUUUCAGUUUCCNN 4103  235-253GAAACUGAAAAACAGAGUANN 4004 UACUCUGUUUUUCAGUUUCNN 4104  346-364GCUAGAAAAUCAGCUUUUANN 4005 UAAAAGCUGAUUUUCUAGCNN 4105  490-508CGGGUCUGCUGAGUCCGCANN 4006 UGCGGACUCAGCAGACCCGNN 4106  700-718CUCCCAGAGGUCUACCCAGNN 4007 CUGGGUAGACCUCUGGGAGNN 4107 1715-1733UUUACUAUGUAAAUGCUUGNN 4008 CAAGCAUUUACAUAGUAAANN 4108  734-752UACCAGCCUCCCUUUCUCUNN 4009 AGAGAAAGGGAGGCUGGUANN 4109  773-791CCAAGCUGGAAGCCAUUAANN 4010 UUAAUGGCUUCCAGCUUGGNN 4110  778-796CUGGAAGCCAUUAAUGAACNN 4011 GUUCAUUAAUGGCUUCCAGNN 4111  779-797UGGAAGCCAUUAAUGAACUNN 4012 AGUUCAUUAAUGGCUUCCANN 4112 1774-1792UCCAUUUAUUUAAAACUACNN 4013 GUAGUUUUAAAUAAAUGGANN 4113  704-722CAGAGGUCUACCCAGAAGGNN 4014 CCUUCUGGGUAGACCUCUGNN 4114 1716-1734UUACUAUGUAAAUGCUUGANN 4015 UCAAGCAUUUACAUAGUAANN 4115 1713-1731AAUUUACUAUGUAAAUGCUNN 4016 AGCAUUUACAUAGUAAAUUNN 4116  768-786AUCAGCCAAGCUGGAAGCCNN 4017 GGCUUCCAGCUUGGCUGAUNN 4117 1129-1147ACUUUUGCCAAUGAACUUUNN 4018 AAAGUUCAUUGGCAAAAGUNN 4118  389-407GUUGAGAACCAGGAGUUAANN 4019 UUAACUCCUGGUUCUCAACNN 4119  701-719UCCCAGAGGUCUACCCAGANN 4020 UCUGGGUAGACCUCUGGGANN 4120  706-724GAGGUCUACCCAGAAGGACNN 4021 GUCCUUCUGGGUAGACCUCNN 4121  707-725AGGUCUACCCAGAAGGACCNN 4022 GGUCCUUCUGGGUAGACCUNN 4122  727-745AGUUCCUUACCAGCCUCCCNN 4023 GGGAGGCUGGUAAGGAACUNN 4123  733-751UUACCAGCCUCCCUUUCUCNN 4024 GAGAAAGGGAGGCUGGUAANN 4124  736-754CCAGCCUCCCUUUCUCUGUNN 4025 ACAGAGAAAGGGAGGCUGGNN 4125  738-756AGCCUCCCUUUCUCUGUCANN 4026 UGACAGAGAAAGGGAGGCUNN 4126  743-761CCCUUUCUCUGUCAGUGGGNN 4027 CCCACUGACAGAGAAAGGGNN 4127  769-787UCAGCCAAGCUGGAAGCCANN 4028 UGGCUUCCAGCUUGGCUGANN 4128  772-790GCCAAGCUGGAAGCCAUUANN 4029 UAAUGGCUUCCAGCUUGGCNN 4129  774-792CAAGCUGGAAGCCAUUAAUNN 4030 AUUAAUGGCUUCCAGCUUGNN 4130  231-249GGAGGAAACUGAAAAACAGNN 4031 CUGUUUUUCAGUUUCCUCCNN 4131  233-251AGGAAACUGAAAAACAGAGNN 4032 CUCUGUUUUUCAGUUUCCUNN 4132  735-753ACCAGCCUCCCUUUCUCUGNN 4033 CAGAGAAAGGGAGGCUGGUNN 4133  737-755CAGCCUCCCUUUCUCUGUCNN 4034 GACAGAGAAAGGGAGGCUGNN 4134  739-757GCCUCCCUUUCUCUGUCAGNN 4035 CUGACAGAGAAAGGGAGGCNN 4135  740-758CCUCCCUUUCUCUGUCAGUNN 4036 ACUGACAGAGAAAGGGAGGNN 4136  746-764UUUCUCUGUCAGUGGGGACNN 4037 GUCCCCACUGACAGAGAAANN 4137  770-788CAGCCAAGCUGGAAGCCAUNN 4038 AUGGCUUCCAGCUUGGCUGNN 4138   26-44GCUAUGGUGGUGGUGGCAGNN 4039 CUGCCACCACCACCAUAGCNN 4139   27-45CUAUGGUGGUGGUGGCAGCNN 4040 GCUGCCACCACCACCAUAGNN 4140  732-750CUUACCAGCCUCCCUUUCUNN 4041 AGAAAGGGAGGCUGGUAAGNN 4141

1. A dual targeting siRNA agent comprising a first dsRNA targeting aPCSK9 gene and a second dsRNA targeting a second gene, wherein the firstdsRNA and the second dsRNA are linked with a covalent linker. 2.(canceled)
 3. The dual targeting siRNA agent of claim 1, wherein thesecond gene is selected from the group consisting of XBP-1, PCSK9,PCSK5, ApoC3, SCAP, and MIG12.
 4. (canceled)
 5. The dual targeting siRNAagent of claim 1, wherein the first dsRNA comprises at least 15contiguous nucleotides of an antisense strand of one of Tables 1, 2, or4-8, or comprises an antisense strand of one of Tables 1, 2, or 4-8, orcomprises a sense strand and an antisense strand of one of Tables 1, 2,or 4-8.
 6. The dual targeting siRNA agent of claim 1, wherein the firstdsRNA comprises AD-9680 or AD-10792.
 7. The dual targeting siRNA agentof claim 1, wherein the second dsRNA comprises at least 15 contiguousnucleotides of an antisense strand of one of Tables 3 or 9-13, orcomprises an antisense strand of one of Tables 3 or 9-13, or comprises asense strand and an antisense strand of one of Tables 3 or 9-13.
 8. Thedual targeting siRNA agent of claim 1, wherein the second dsRNAcomprises AD-18038.
 9. The dual targeting siRNA agent of claim 1,wherein the first and second dsRNA comprises at least one modifiednucleotide.
 10. The dual targeting siRNA agent of claim 9, wherein themodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group.
 11. The dual targeting siRNA agent of claim 9,wherein the modified nucleotide is chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 12. Thedual targeting siRNA agent of claim 1, wherein each strand of each dsRNAis 19-23 bases in length.
 13. The dual targeting siRNA agent of claim 1,wherein the first and second dsRNAs are linked with a disulfide linker.14. The dual targeting siRNA agent of claim 1, wherein the covalentlinker links the sense strand of the first dsRNA to the sense strand ofthe second dsRNA.
 15. The dual targeting siRNA agent of claim 1, whereinthe covalent linker links the antisense strand of the first dsRNA to theantisense strand of the second dsRNA.
 16. The dual targeting siRNA agentof claim 1, further comprising a ligand.
 17. (canceled)
 18. (canceled)19. A pharmaceutical composition comprising the dual targeting siRNAagent of claim 1 and a pharmaceutical carrier.
 20. The pharmaceuticalcomposition of claim 19, wherein the pharmaceutical carrier is a lipidformulation. 21.-23. (canceled)
 24. A method of inhibiting expression ofthe PCSK9 gene and a second gene in a cell, the method comprising (a)introducing into the cell the dual targeting siRNA agent of claim 1; and(b) maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of the PCSK9 gene and thesecond gene, thereby inhibiting expression of the PCSK9 gene and thesecond gene in the cell.
 25. A method of treating a disorder mediated byPCSK9 expression or of reducing total serum cholesterol in a subjectcomprising administering to a subject in need of such treatment atherapeutically effective amount of the pharmaceutical composition ofclaim
 19. 26.-30. (canceled)